Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector

ABSTRACT

Systems and method for detecting optical signals, and for discriminating optical signals emitted by an emission moiety that is excited by an associated excitation signal from background signals and other optical noise, employing digital techniques for determining the portion of a detected optical signal having a modulation frequency corresponding to a modulation of the associated excitation signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application under 35 U.S.C. §§ 120, 121of U.S. patent application Ser. No. 13/404,437, filed Feb. 24, 2012, nowU.S. Pat. No. 8,718,948, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/446,280, filed Feb. 24,2011, the disclosures of which are hereby incorporated by reference intheir entirety.

BACKGROUND

Field of the Invention

The present invention relates to systems and methods for performingmultiple diagnostic assays simultaneously, and more particularly tosystems and methods for distinguishing different measured emissionsignals based on the modulation frequencies of the correspondingexcitation signals that generated the emission signals.

Background of the Invention

None of the references described or referred to herein are admitted tobe prior art to the claimed invention.

Diagnostic assays are widely used in clinical diagnosis and healthscience research to detect or quantify the presence or amount ofbiological antigens, cell or genetic abnormalities, disease states, anddisease-associated pathogens or genetic mutations in an organism orbiological sample. Where a diagnostic assay permits quantification,practitioners may be better able to calculate the extent of infection ordisease and to determine the state of a disease over time. Diagnosticassays are frequently focused on the detection of chemicals, proteins orpolysaccharides antigens, nucleic acids, biopolymers, cells, or tissueof interest. A variety of assays may be employed to detect thesediagnostic indicators.

Nucleic acid-based assays, in particular, generally include multiplesteps leading to the detection or quantification of one or more targetnucleic acid sequences in a sample. The targeted nucleic acid sequencesare often specific to an identifiable group of proteins, cells, tissues,organisms, or viruses, where the group is defined by at least one sharedsequence of nucleic acid that is common to members of the group and isspecific to that group in the sample being assayed. A variety of nucleicacid-based detection methods are fully described by Kohne, U.S. Pat. No.4,851,330, and Hogan, U.S. Pat. No. 5,541,308.

Detection of a targeted nucleic acid sequence frequently requires theuse of a nucleic acid molecule having a nucleotide base sequence that issubstantially complementary to at least a portion of the targetedsequence or its amplicon. Under selective assay conditions, the probewill hybridize to the targeted sequence or its amplicon in a mannerpermitting a practitioner to detect the presence of the targetedsequence in a sample. Techniques of effective probe preparation areknown in the art. In general, however, effective probes are designed toprevent non-specific hybridization with itself or any nucleic acidmolecule that will interfere with detecting the presence of the targetedsequence. Probes may include, for example, a label capable of detection,where the label is, for example, a radiolabel, a fluorophore orfluorescent dye, biotin, an enzyme, a chemiluminescent compound, oranother type of detectable signal known in the art.

Because the probe hybridizes to the targeted sequence or its amplicon ina manner permitting detection of a signal indicating the presence of thetargeted sequence in a sample, the strength of the signal isproportional to the amount of target sequence or its amplicon that ispresent. Accordingly, by periodically measuring, during an amplificationprocess, a signal indicative of the presence of amplicon, the growth ofamplicon overtime can be detected. Based on the data collected duringthis “real-time” monitoring of the amplification process, the amount ofthe target nucleic acid that was originally in the sample can beascertained. Systems and methods for real time detection and forprocessing real time data to ascertain nucleic acid levels aredescribed, for example, in Lair, et al., U.S. Pat. No. 7,932,081,“Signal measuring system for conducting real-time amplification assays,”the disclosure of which is hereby incorporated by reference.

To detect different nucleic acids of interest in a single assay,different probes configured to hybridize to different nucleic acids,each of which may provide detectibly different signals can be used. Forexample, different probes configured to hybridize to different targetscan be formulated with fluorophores that fluoresce at a predeterminedwavelength when exposed to excitation light of a prescribed excitationwavelength. Assays for detecting different target nucleic acids can beperformed in parallel by alternately exposing the sample material todifferent excitation wavelengths and detecting the level of fluorescenceat the wavelength of interest corresponding to the probe for each targetnucleic acid during the real-time monitoring process. Parallelprocessing can be performed using different signal detecting devicesconstructed and arranged to periodically measure signal emissions duringthe amplification process, and with different signal detecting devicesbeing configured to generate excitation signals of different wavelengthsand to measure emission signals of different wavelengths.

Occasionally, however, the excitation and emission wavelengths for onefluorophore will overlap the excitation and emission wavelengths ofanother fluorophore. In such circumstances, it becomes a challenge toensure that a measured signal is entirely due to an emission of thefluorophore of interest, excited by an intended excitation signal. Such“optical crosstalk” can take a number of forms. For signal detectingdevices configured to measure emissions from samples held in closelyadjacent reaction receptacles, crosstalk can occur when one channeldetects the excitation light from another channel (of the same ordifferent signal detector) or when one signal detecting device picks upemission light from a receptacle that is excited by a different signaldetecting device. In addition, crosstalk can occur when an excitationsignal for a particular dye color excites a dye of a color that is notintended for that signal detector. Furthermore, crosstalk can occur whenan emission signal for a particular dye color excites a dye of a colorthat is not intended for that signal detector.

Synchronous detection is a means to reduce some forms of crosstalk, aswell as optical noise due to, for example, ambient light. Synchronousdetection creates a narrow bandwidth filter that is sensitive to anarrow range of frequencies in the emission signal centered at amodulation frequency of the excitation signal. The excitation signalfrom the signal detecting device is demodulated at the modulationfrequency, and a fluorescence detection circuit is configured to detectthe frequency of the measured emission signal and to reject portions ofthe signal having a frequency that is inconsistent with the excitationfrequency. Such a circuit-based “analog demodulator” is described, forexample, in Lair, et al., U.S. Pat. No. 7,932,081.

SUMMARY OF THE INVENTION

The systems and methods of the present invention provide improvedprocedures for performing synchronous detection as well as improvedprocedures and mechanisms for exploiting synchronous detection forlimiting or avoiding crosstalk.

Accordingly, aspects of the invention are embodied in a method fordetecting multiple different optical signals emitted from the contentsof each of two or more receptacles, each different optical signalindicating the presence of a different analyte of interest oramplification products thereof. The method comprises formingamplification products of an analyte of interest within each receptacle,and, while forming the amplification products, directing an excitationsignal at each receptacle. The excitation signal has a predeterminedexcitation wavelength that excites an emission moiety, which emits anemission signal that is associated with the excitation wavelength andhas a predetermined emission wavelength, and each excitation signal hasa predetermined excitation frequency. With a signal detecting device, anoptical signal, including an emission signal emitted from eachreceptacle, is detected and detection data from the detected signal isgenerated. The detection data is digitized by a computer-implementedalgorithm. An amplitude of the detection data at the predeterminedexcitation frequency is determined from the digitized data by acomputer-implemented algorithm to ascertain the portion of the detectedsignal that corresponds to the associated emission signal. The steps ofdirecting the excitation signal, detecting an optical signal, digitizingthe detection data, and detecting the amplitude of the detection data atthe predetermined excitation frequency are repeated for each of themultiple different optical signals to be detected. The predeterminedexcitation frequency for an excitation signal associated with theemission signal of one emission moiety is different from thepredetermined excitation frequency for at least one other emissionmoiety in the receptacle.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein detecting an optical signalcomprises measuring an intensity of an optical signal at each of aplurality of discrete times, and wherein the measured intensity of theoptical signal at each discrete time comprises an average intensitymeasured over a range of time including the discrete time.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein three or more differentoptical signals are detected from each of the receptacles.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the excitation frequenciesare the same for two or more, but less than all, of the optical signalsto be detected.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the emission signal is afluorescent emission.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the emission moietycomprises a fluorescent dye, and each different optical signal isgenerated by a different fluorescent dye contained in the receptacle.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the at least one opticalsignal comprises first, second, and third optical signals. The firstoptical signal comprises a first range of optical wavelengths, thesecond optical signal comprises a second range of optical wavelengths,and the third optical signal comprises a third range of opticalwavelengths. The spectra of the first and second range of opticalwavelengths at least partially overlap each other, the spectra of thesecond and third range of optical wavelengths at least partially overlapeach other, but the spectra of the first and third range of opticalwavelengths do not overlap each other. The excitation frequencies of theexcitation signals associated with the first and third optical signalsare the same, and the excitation frequency of the excitation signalassociated with the second optical signal is different from theexcitation frequencies of the excitation signals associated with thefirst and third optical signals.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the two or more receptaclesare arranged in a linear, side-by-side arrangement and wherein thepredetermined excitation frequencies for adjacent receptacles aredifferent. In some embodiments, the predetermined excitation frequenciesfor alternate receptacles are the same.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein detecting the amplitude ofthe detection data at the predetermined excitation frequency comprisesexecuting a Goertzel signal processing technique.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, which also includes the step ofexecuting a computer-implemented algorithm for determining, from datarelating to the portion of the detected signal that corresponds to theassociated emission signal, the amount of an analyte present within eachreceptacle.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the detection data isdigitized at a frequency that is at least twice the excitation frequencyof an associated excitation signal.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the excitation frequency iswithin the range of 200-350 Hz, and the digitization frequency is about4 kHz. Further aspects of the invention are embodied in the method ofdetecting multiple different optical signals, wherein the excitationfrequency is within the range of 100 Hz-1 kHz, and the digitizationfrequency is within the range of 2 kHz-8 kHz. Further aspects of theinvention are embodied in the method of detecting multiple differentoptical signals, wherein the excitation frequency is within the range of10 Hz-5 kHz, and the digitization frequency is within the range of 1kHz-1 GHz.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, which further includes the steps offorming amplification products of a control analyte and performing thesteps of directing the excitation signal, detecting an optical signal,digitizing the detection data, and detecting the amplitude of thedetection data at the predetermined excitation frequency for the controlanalyte.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein the control analytecomprises a known analyte unrelated to the analyte of interest.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, which further includes adding acontrol probe having specificity for the control analyte to eachreceptacle, wherein the control probe comprises an emission moiety thatemits an emission signal having an emission wavelength that is differentfrom the emission wavelength of the emission moiety for the analyte ofinterest.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, which further includes executing acomputer-implemented algorithm for determining from data relating to theportion of the detected signal that corresponds to the associatedemission signal the amount of the control analyte.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, which further includes alternatelyexposing the contents of the receptacle to light energy at differentexcitation wavelengths and detecting the presence or absence of a signalhaving the emission wavelength of the emission moiety of the controlprobe as in indication of the successful formation of amplificationproducts.

Further aspects of the invention are embodied in the method of detectingmultiple different optical signals, wherein each receptacle isreleasably fixed within the side-by-side arrangement.

Aspects of the invention are also embodied in a system for detecting anoptical signal emitted from a receptacle. The system comprises anexcitation device and a signal detecting device. The excitation deviceis constructed and arranged to generate an excitation signal. Theexcitation signal has a predetermined excitation wavelength and apredetermined excitation frequency and excites an emission moiety, whichemits an emission signal that is associated with the excitationwavelength and has a predetermined emission wavelength. The excitationdevice is further constructed and arranged to direct the excitationsignal at the receptacle. The signal detecting device is constructed andarranged to detect an optical signal, including an emission signalemitted from the receptacle, and to store detection data relating to thedetected signal on a computer-readable medium. The system furtherincludes a processor for executing an algorithm to digitize the storeddetection data and a processor for executing an algorithm fordetermining, from the digitized detection data, an amplitude of theemission signal at the predetermined excitation frequency to ascertainthe portion of the detected signal corresponding to the associatedemission signal.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, which furtherincludes a processor for executing an algorithm for determining theamount of an analyte present within the receptacle based on datarelating to the portion of the detected signal corresponding to theassociated emission signal.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, which furtherincludes a plurality of excitation devices in fixed positions withrespect to each other and a plurality of signal detecting devices infixed positions with respect to each other. Each excitation device isconstructed and arranged to (a) generate an excitation signal having apredetermined excitation wavelength and a predetermined excitationfrequency that excites an emission moiety that emits an associatedemission signal having a predetermined emission wavelength and (b)direct the excitation signal at a different receptacle. Each signaldetecting device is constructed and arranged to detect an emissionsignal emitted from a receptacle and to store detection data relating tothe detected signal on a computer-readable medium. Each excitationdevice corresponds with an associated detecting device such that thegenerated excitation signal and detected emission signal of eachassociated excitation and detecting device are directed at, and emittedfrom, an associated receptacle. The processor is configured to digitizethe stored detection data associated with each signal detecting device,and the processor is configured to determine from the digitizeddetection data associated with each signal detecting device an amplitudeof the emission signal at the associated predetermined excitationfrequency and to ascertain the portion of the associated detected signalthat corresponds to the associated emission signal.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, wherein thedifferent associated receptacles comprise a plurality of receptaclesarranged in a linear, side-by-side arrangement and wherein theassociated predetermined excitation frequencies for adjacent receptaclesare different.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, wherein theassociated predetermined excitation frequencies for alternatereceptacles are the same.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, which furtherincludes a receptacle moving apparatus constructed and arranged to movethe different associated receptacles relative to the plurality ofexcitation and detecting devices.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, which includestwo or more sets of the plurality of excitation and associated detectingdevices, wherein each set is spatially distinct from each other setwithin the system.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, which includesthree to six sets of the plurality of excitation and associateddetecting devices.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, wherein each setof the plurality of excitation and associated detecting devices isarranged radially around a central axis.

Further aspects of the invention are embodied in the system fordetecting an optical signal emitted from a receptacle, wherein eachreceptacle is releasably fixed within the side-by-side arrangement.

Aspects of the invention are also embodied in a method for detecting anoptical signal emitted from the contents of a receptacle in the presenceof multiple, different optical signals originated from the samereceptacle or a different receptacle. The method comprises formingamplification products of an analyte of interest within the receptacle,and, while forming the amplification products, directing an excitationsignal at the receptacle. The excitation signal has a predeterminedexcitation wavelength that excites an emission moiety, which emits anemission signal that is associated with the excitation wavelength andhas a predetermined emission wavelength, and the excitation signal has apredetermined excitation frequency. With a signal detecting device, anoptical signal, including an emission signal emitted from thereceptacle, is detected, and detection data from the detected signal isgenerated. The detection data is digitized using a computer-implementedalgorithm. An amplitude of the detection data at the predeterminedexcitation frequency is determined from the digitized detection data bya computer-implemented algorithm to ascertain the portion of thedetected signal that corresponds to the associated emission signal.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,which further includes detecting a second optical signal emitted fromthe contents of the receptacle. While forming the amplificationproducts, a second excitation signal is directed at the receptacle, thesecond excitation signal having a second predetermined excitationwavelength that excites an emission moiety, which emits a secondemission signal that is associated with the second excitation wavelengthand has a predetermined second emission wavelength, wherein the secondexcitation signal has a predetermined second excitation frequency. Withthe same or a different signal detecting device, an optical signalincluding an emission signal emitted from the receptacle is detected andsecond detection data from the detected signal is generated. The seconddetection data is digitized by a computer-implemented algorithm. Anamplitude of the second detection data at the predetermined secondexcitation frequency is determined from the second digitized detectiondata by a computer-implemented algorithm to ascertain the portion of thedetected signal that corresponds to the associated second emissionsignal.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein the excitation frequencies are different for at least twooptical signals to be detected.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein the emission signal is a fluorescent emission.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein the emission moiety comprises a fluorescent dye.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein first, second, and third optical signals are detected from thereceptacle. The first optical signal comprises a first range of opticalwavelengths, the second optical signal comprises a second range ofoptical wavelengths, and the third optical signal comprises a thirdrange of optical wavelengths. The spectra of the first and second rangeof optical wavelengths at least partially overlap each other, thespectra of the second and third range of optical wavelengths at leastpartially overlap each other, but the spectra of the first and thirdrange of optical wavelengths do not overlap each other. The excitationfrequencies of the excitation signals associated with the first andthird optical signals are the same and the excitation frequency of theexcitation signal associated with the second optical signal is differentfrom the excitation frequencies of the excitation signals associatedwith the first and third optical signals.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein determining the amplitude of the detection data at thepredetermined frequency comprises executing a Goertzel signal processingtechnique.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,which further includes executing a computer-implemented algorithm fordetermining, from data relating to the portion of the detected signalthat corresponds to the associated emission signal, the amount of ananalyte present within the receptacle.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein the detection data is digitized at a frequency that is at leasttwice the excitation frequency of the associated excitation signal.

Further aspects of the invention are embodied in the method fordetecting an optical signal emitted from the contents of a receptacle,wherein the excitation frequency is within the range of 200-350 Hz, andthe digitization frequency is about 4 kHz. Further aspects of theinvention are embodied in the method of detecting an optical signalemitted from the contents of a receptacle, wherein the excitationfrequency is within the range of 100 Hz-1 kHz, and the digitizationfrequency is within the range of 2 kHz-8 kHz. Further aspects of theinvention are embodied in the method of detecting an optical signalemitted from the contents of a receptacle, wherein the excitationfrequency is within the range of 10 Hz-5 kHz, and the digitizationfrequency is within the range of 1 kHz-1 GHz.

These and other features, aspects, and advantages of the presentinvention will become apparent to those skilled in the art afterconsidering the following detailed description, appended claims andaccompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a reaction receptacle in the form of amultiple receptacle device unit employed in combination with anapparatus embodying aspects of the present invention.

FIG. 2 is a side elevation of a contact-limiting pipette tiplet employedin combination with an instrument for performing a magnetic separationprocedure and carried on the multiple receptacle device shown in FIG. 1.

FIG. 3 is an enlarged bottom view of a portion of the multiplereceptacle device, viewed in the direction of arrow “60” in FIG. 1.

FIG. 4 is an exploded perspective view of an incubator configured tohold a plurality of receptacles while subjecting the reactionreceptacles to prescribed temperature conditions and including signaldetectors for detecting signals emitted by the contents of the reactionreceptacles during an incubation process.

FIG. 5 is a bottom plan view of a receptacle carrier carousel of theincubator.

FIG. 6 is a perspective view of assembled components of a receptaclecarrier carousel of the incubator and a circulating fan for generatingairflow within the incubator.

FIG. 7 is a perspective view of a bottom wall of the incubator housing,a portion of the receptacle carrier, and a receptacle carrier driveassembly.

FIG. 8 is a perspective view of a receptacle divider of the receptaclecarrier.

FIG. 9 is a perspective view of the receptacle divider from an oppositeside of the divider.

FIG. 10 is a partial perspective view of components of the receptaclecarrier of the incubator including a receptacle present sensor fordetecting the presence of reaction receptacles on the receptaclecarrier.

FIG. 11 is a partial perspective view of the incubator and a receptacletransport mechanism adapted to place reaction receptacles into theincubator and remove reaction receptacles from the incubator.

FIG. 12 is a partial perspective view of a portion of the incubatorincluding the incubator floor, signal detectors disposed beneath theincubator floor, and reaction receptacles disposed in signal detectingpositions with respect to the signal detectors.

FIG. 13 is a perspective view of a signal detector for use inconjunction with the present invention.

FIG. 14 is a bottom plan view of the signal detector.

FIG. 15 is a side cross-sectional view of the signal detector takenalong the line 15-15 in FIG. 14.

FIG. 16 is an exploded perspective view of the signal detector.

FIG. 17 is a graph showing excitation spectra of preferred amplificationdetection dyes.

FIG. 18 is a graph showing emission spectra of preferred amplificationdetection dyes.

FIG. 19 is a flow chart showing the protocols of a preferred real-timeamplification assay in accordance with aspects of the present invention.

FIG. 20 is a flow chart showing an analyte quantification process.

FIG. 21 is a time plot of real-time fluorometer data;

FIG. 22 is a plot showing a method for fitting a curve to real-timefluorometer data and using the fit to determine a threshold time.

FIG. 23 is a flow chart illustrating an algorithm for performing phasesynchronous detection of an emission signal from the contents of areaction receptacle to detect the frequency component of the emissionsignal that is due to the correct excitation signal.

FIG. 24 is a block diagram schematically illustrating excitation anddetection architecture.

FIG. 25 is a block diagram schematically illustrating an arrangement ofdetection circuitry embodying aspects of the invention.

FIGS. 26A and 26B are two parts of a circuit diagram illustrating afluorometer detection circuit embodying aspects of the invention.

FIG. 27 is a block diagram schematically illustrating an arrangement ofexcitation circuitry embodying aspects of the invention.

FIG. 28 is a circuit diagram illustrating a fluorometer excitationcircuit embodying aspects of the invention.

FIG. 29 is scheduling diagram showing movement of reaction receptacles,measurement of emission signals from the reaction receptacles, andprocessing of the signals measured from the reaction receptacles.

FIG. 30 shows the bandwidths of excitation and emission filters used inoptical signal detectors for detecting different fluorescent dyes.

FIG. 31 is a graph showing excitation and emission fluorescence spectrafor FAM, HEX, and ROX dyes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Multiple ReceptacleDevices

Referring to FIG. 1, a reaction receptacle in the form of a multiplereceptacle device (“MRD”) 160 comprises a plurality of individualreceptacle vessels, or reaction tubes, 162, preferably five. Thereceptacle vessels 162, preferably in the form of cylindrical tubes withopen top ends and closed bottom ends, are connected to one another by aconnecting rib structure 164 which defines a downwardly facing shoulderextending longitudinally along either side of the MRD 160.

The MRD 160 is preferably formed from injection molded polypropylene,such as those sold by Montell Polyolefins, of Wilmington, Del., productnumber PD701NW or Huntsman, product number P5M6K-048. In an alternativeembodiment, the receptacle vessels 162 of the MRD are releasably fixedwith respect to each other by means such as, for example, a sample tuberack.

An arcuate shield structure 169 is provided at one end of the MRD 160.An MRD manipulating structure 166 extends from the shield structure 169.The manipulating structure is adapted to be engaged by a transportmechanism for moving the MRD 160 between different components of adiagnostic analyzer. An exemplary transport mechanism that is compatiblewith the MRD 160 is described in U.S. Pat. No. 6,335,166, the disclosureof which is hereby incorporated by reference. The MRD manipulatingstructure 166 comprises a laterally extending plate 168 extending fromshield structure 169 with a vertically extending piece 167 on theopposite end of the plate 168. A gusset wall 165 extends downwardly fromlateral plate 168 between shield structure 169 and vertical piece 167.

As shown in FIG. 3, the shield structure 169 and vertical piece 167 havemutually facing convex surfaces. The MRD 160 may be engaged by atransport mechanism and other components, by moving an engaging memberlaterally (in the direction “A”) into the space between the shieldstructure 169 and the vertical piece 167. The convex surfaces of theshield structure 169 and vertical piece 167 provide for wider points ofentry for an engaging member undergoing a lateral relative motion intothe space.

A label-receiving structure 174 having a flat label-receiving surface175 is provided on an end of the MRD 160 opposite the shield structure169 and MRD manipulating structure 166. Human and/or machine-readablelabels, such as scannable bar codes, can be placed on the surface 175 toprovide identifying and instructional information on the MRD 160.

The MRD 160 preferably includes tiplet holding structures 176 adjacentthe open mouth of each respective receptacle vessel 162. Each tipletholding structure 176 provides a cylindrical orifice within which isreceived conduit that is adapted to be placed onto the end of anaspirating tube, such as contact-limiting tiplet 170. Exemplaryconstruction and function of the tiplet 170 is described below. Eachholding structure 176 is constructed and arranged to frictionallyreceive a tiplet 170 in a manner that prevents the tiplet 170 fromfalling out of the holding structure 176 when the MRD 160 is inverted,but permits the tiplet 170 to be removed from the holding structure 176when engaged by a pipette.

Referring to FIG. 2, the tiplet 170 comprises a generally cylindricalstructure having a peripheral rim flange 177 and an upper collar 178 ofgenerally larger diameter than a lower portion 179 of the tiplet 170.The tiplet 170 is preferably formed from conductive polypropylene. Whenthe tiplet 170 is inserted into an orifice of a holding structure 176,the flange 177 contacts the top of structure 176 and the collar 178provides a snug but releasable interference fit between the tiplet 170and the holding structure 176. Alternatively, each holding structure 176may be configured to loosely receive a tiplet 170 so that the tiplet ismore easily removed from the holding structure when engaged by apipette.

Further details regarding the MRD 160 may be found in U.S. Pat. No.6,086,827, the disclosure of which is hereby incorporated by reference.Though a particular configuration of MRD 160 is exemplified, one ofskill in the art would appreciate that a variety of configurations ofsingle or multiple receptacle devices may be utilized according to thepresent invention.

Specimen Preparation Procedure

In an exemplary embodiment, samples are prepared for a magneticseparation procedure by dispensing an amount of target capture reagent,preferably mag-oligo reagent, e.g., by an automated, robotic pipettingapparatus, into each of the receptacle vessels 162 of the MRD 160. Thetarget capture reagent includes a support material able to bind to andimmobilize a target analyte. The support material preferably comprisesmagnetically responsive particles. The amount dispensed into eachreceptacle vessel 162 is typically 100-500 μl. Next, an amount of samplematerial is dispensed into each of the receptacle vessels 162 of the MRD160 containing target capture reagent. A different sample may bedispensed into each of the five receptacle vessels 162, or the samesample may dispensed into two or more of the receptacle vessels 162. Amagnetic separation procedure includes the steps of exposing thecontents of the receptacle vessel to a magnetic field to draw themagnetically-responsive particles to a side of the receptacle vessel andout of suspension, withdrawing the fluid contents of the receptaclevessel (e.g., by vacuum aspiration) while holding themagnetically-responsive particles out of suspension, removing themagnetic field, and adding a wash solution to the receptacle vessel tore-suspend the magnetically-responsive particles. These steps may berepeated one or more times. A suitable magnetic separation washprocedure is described in Lair et al., U.S. Pat. No. 8,008,066, thedisclosure of which is hereby incorporated by reference.

Real-Time Amplification Assays

Real-time amplification assays can be used to determine the presence andamount of a target nucleic acid in a sample which, by way of example, isderived from a pathogenic organism or virus. By determining the quantityof a target nucleic acid in a sample, a practitioner can approximate theamount or load of the organism or virus in the sample. In oneapplication, a real-time amplification assay may be used to screen bloodor blood products intended for transfusion for bloodborne pathogens,such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV).In another application, a real-time assay may be used to monitor theefficacy of a therapeutic regimen in a patient infected with apathogenic organism or virus, or that is afflicted with, or suspected ofbeing afflicted with, a disease characterized by aberrant or mutant geneexpression. Real-time amplification assays may also be used fordiagnostic purposes, as well as in gene expression determinations. In apreferred application of the present invention discussed above, thepresence of an organism, virus, or gene of interest is determined usinga probe which, under the particular conditions of use, exhibitsspecificity in a sample for a target nucleic acid sequence derived fromthe organism, virus, or gene of interest (i.e., contained within targetnucleic acid obtained from the organism, virus, or gene of interest, oran amplification product thereof). To exhibit specificity, a probegenerally has a nucleotide base sequence that is substantiallycomplementary to at least a portion of the target or its complement suchthat, under selective assay conditions, the probe will detectablyhybridize to the target sequence or its complement but not to anynon-target nucleic acids that may be present in the sample.

Aspects of the present invention relate to systems and methods forperforming “real-time” amplification assays which are distinguishablefrom “end-point” amplification assays. In “end-point” amplificationassays, the presence of amplification products containing the targetsequence or its complement is determined at the conclusion of anamplification procedure. The determination may occur in a detectionstation that may be located externally to the incubator in which theamplification reactions occur. In contrast, in “real-time” amplificationassays, the amount of amplification products containing the targetsequence or its complement is determined during an amplificationprocedure. In the real-time amplification assay, the concentration of atarget nucleic acid can be determined using data acquired by makingperiodic measurements of signals that are functions of the amount ofamplification product in the sample containing the target sequence, orits complement, and calculating the rate at which the target sequence isbeing amplified from the acquired data.

For real-time amplification assays, the probes are preferablyunimolecular, self-hybridizing probes having a pair of interactinglabels that interact and thereby emit different signals, depending onwhether the probes are in a self-hybridized state or hybridized to thetarget sequence or its complement. See, e.g., Diamond et al.,“Displacement Polynucleotide Assay Method and Polynucleotide ComplexReagent Therefor,” U.S. Pat. No. 4,766,062; Tyagi et al., “DetectablyLabeled Dual Conformation Oligonucleotide Probes, Assays and Kits,” U.S.Pat. No. 5,925,517; Tyagi et al., “Nucleic Acid Detection Probes HavingNon-FRET Fluorescence Quenching and Kits and Assays Including SuchProbes,” U.S. Pat. No. 6,150,097; and Becker et al., “MolecularTorches,” U.S. Pat. No. 6,361,945. Other probes are contemplated for usein the present invention, including complementary, bimolecular probes,probes labeled with an intercalating dye and the use of intercalatingdyes to distinguish between single-stranded and double-stranded nucleicacids. See, e.g., Morrison, “Competitive Homogenous Assay,” U.S. Pat.No. 5,928,862; Higuchi, “Homogenous Methods for Nucleic AcidAmplification and Detection,” U.S. Pat. No. 5,994,056; and Yokoyama etal., “Method for Assaying Nucleic Acid,” U.S. Pat. No. 6,541,205.Examples of interacting labels include enzyme/substrate,enzyme/cofactor, luminescent/quencher, luminescent/adduct, dye dimersand Förrester energy transfer pairs. Methods and materials for joininginteracting labels to probes for optimal signal differentiation aredescribed in the above-cited references.

In a preferred real-time amplification assay, the interacting labelsinclude a fluorescent moiety, or other emission moiety, and a quenchermoiety, such as, for example, 4-(4-dimethylaminophenylazo) benzoic acid(DABCYL). The fluorescent moiety emits light energy (i.e., fluoresces)at a specific emission wavelength when excited by light energy at anappropriate excitation wavelength. When the fluorescent moiety and thequencher moiety are held in close proximity, light energy emitted by thefluorescent moiety is absorbed by the quencher moiety. But when a probehybridizes to nucleic acid present in the sample, the fluorescent andquencher moieties are separated from each other and light energy emittedby the fluorescent moiety can be detected. Fluorescent moieties whichare excited and emit at different and distinguishable wavelengths can becombined with different probes. The different probes can be added to asample, and the presence and amount of target nucleic acids associatedwith each probe can be determined by alternately exposing the sample tolight energy at different excitation wavelengths and measuring the lightemission from the sample at the different wavelengths corresponding tothe different fluorescent moieties.

In one example of a multiplex, real-time amplification assay, thefollowing may be added to a sample prior to initiating the amplificationreaction: a first probe having a quencher moiety and a first fluorescentdye (having an excitation wavelength λ_(ex1) and emission wavelengthλ_(em1)) joined to its 5′ and 3′ ends and having specificity for anucleic acid sequence derived from HCV; a second probe having a quenchermoiety and a second fluorescent dye (having an excitation wavelengthλ_(ex2) and emission wavelength λ_(em2)) joined to its 5′ and 3′ endsand having specificity for a nucleic acid sequence derived from HIV Type1 (HIV-1); and a third probe having a quencher moiety and a thirdfluorescent dye (having an excitation wavelength λ_(ex3) and emissionwavelength λ_(em3)) joined to its 5′ and 3′ ends and having specificityfor a nucleic acid sequence derived from West Nile virus (WNV). Aftercombining the probes in a sample with amplification reagents, thesamples can be periodically and alternately exposed to excitation lightat wavelengths λ_(ex1), λ_(ex2), and λ_(ex3), and then measured foremission light at wavelengths, λ_(em1), λ_(em2), and λ_(em3), to detectthe presence (or absence) and amount of all three viruses in the singlesample. The components of an amplification reagent will depend on theassay to be performed, but will generally contain at least oneamplification oligonucleotide, such as a primer, a promoter-primer,and/or a promoter oligonucleotide, nucleoside triphosphates, andcofactors, such as magnesium ions, in a suitable buffer.

Where an amplification procedure is used to increase the amount oftarget sequence, or its complement, present in a sample before detectioncan occur, it is often desirable to include a “control” to ensure thatamplification has taken place and, thereby, to avoid false negatives.Such a control can be a known nucleic acid sequence that is unrelated tothe sequence(s) of interest, or another sequence. A probe (i.e., acontrol probe) having specificity for the control sequence and having aunique fluorescent dye (i.e., the control dye) and quencher combinationis added to the sample, along with one or more amplification reagentsneeded to amplify the control sequence, as well as the targetsequence(s). After exposing the sample to appropriate amplificationconditions, the sample is alternately exposed to light energy atdifferent excitation wavelengths (including the excitation wavelengthfor the control dye) and emission light is detected. Detection ofemission light of a wavelength corresponding to the control dye confirmsthat the amplification was successful (i.e., the control sequence wasindeed amplified), and thus, any failure to detect emission lightcorresponding to the probe(s) of the target sequence(s) is not likelydue to a failed amplification. Conversely, failure to detect emissionlight from the control dye is likely indicative of a failedamplification, thus rendering any results from that assay suspect.

Systems and method for performing real-time amplification assays aredescribed in Lair et al., “System for performing multi-formattedassays,” U.S. Pat. No. 8,008,066, the disclosure of which is herebyincorporated by reference.

In accordance with aspects of the present invention, real-timeamplification assays are performed in an incubator, such as incubator200, features of which are shown in FIGS. 4-12. Incubator 200 is arotary incubator in the sense that MRD's 160 are carried on a rotarycarrier structure (e.g., a carousel) within a housing having acontrolled temperature interior. Incubator 200 includes signaldetectors, or signal detector blocks, 400 attached thereto fordetecting, in a real-time manner, the amplification occurring within thereaction tubes 162 of an MRD 160 carried in the incubator, for example,by measuring the fluorescence emitted by a dye or dyes within eachreaction tube 162 of the MRD 160 when the MRD 160 is illuminated with anexcitation light corresponding to each dye. The incubator 200 can beintegrated into an automated diagnostic analyzer (not shown) that mayinclude one or more receptacle transport mechanisms for placing MRD's160 into the incubator 200 and removing MRD's 160 from the incubator200.

Features of an incubation station, or incubator 200, adapted for use inconjunction with the present invention, are shown in FIGS. 4-12. FIG. 4shows an exploded perspective view of the incubator 200. The incubator200 includes a housing that comprises an outer wall 202, a bottom wall206, and a top wall (not shown), all of which are covered by a thermalinsulating shroud, or hood, 212. The side, bottom and top walls arepreferably formed of aluminum, and the insulating hood is preferablymade from a suitable insulating material, such as polyurethane foam. Areceptacle carrier 242, preferably in the form of a carousel rotatablymounted within the housing, is configured for carrying a plurality ofreaction receptacles. Receptacles, such as MRDs 160, can be insertedinto the receptacle carrier 242 and removed from the receptacle carrier242 through a receptacle opening 204 formed in the sidewall 202.Receptacle opening 204 is covered by the sliding door 216 of a doorassembly 214 (described in more detail below).

One or more signal detectors 400 are disposed beneath the bottom wall206 of the incubator housing and are configured for detecting signalsemitted by the contents of reaction MRDs 160 carried on the receptaclecarrier 242 within the incubator 200. The signal detectors 400 aredescribed in further detail below.

Heat may be generated within the incubator 200 by any suitable means. Inone embodiment, resistive heating elements are disposed on the sidewall202 of the incubator housing. Other suitable heating elements mayinclude, for example, Peltier' thermoelectric heating elements. Theheating elements may be under microprocessor control for maintaining aconstant, desired temperature, and the incubator 200 may further includeone or more temperature-sensing elements for providing temperature levelsignals to the microprocessor controller.

A circulating fan 226 may be positioned within the incubator housing,for example, atop the receptacle carrier 242. In one embodiment, fan 226is an axial fan, as shown, configured for generating airflow through thereceptacle carrier 242 and within the incubator 200.

Further details concerning the construction of the receptacle carrier242 are shown in FIGS. 5 and 6. FIG. 5 is a bottom plan view of thereceptacle carrier 242 with a plurality of MRDs 160 carried thereon.FIG. 6 is a perspective view of a portion of the receptacle carrier 242and showing the fan 226 mounted atop the carrier 242.

Carrier 242 comprises an upper disk 244 and an identical lower disk 256.As shown in FIGS. 5 and 6, the lower disk includes an inner ring 258, anouter ring 260, and an intermediate ring 262 disposed concentricallybetween the inner ring 258 and outer ring 260. Inner radial spokes 266extend between the inner ring 248 and the intermediate ring 262. Outerspokes 264 extend between the intermediate ring 262 and the outer ring260 and are, in this embodiment, configured obliquely with respect to atrue-radial orientation relative to the center of the intermediate ring262 and outer ring 260.

The upper disk 244 has a similar construction, but only outer ring 248and outer spokes 252 are visible in FIG. 6. The upper disk 244 furtherincludes an inner ring, an intermediate ring, and inner spokes, all ofwhich are obstructed from view by the fan 226 in FIG. 6.

The upper disk 244 and the lower disk 256 are secured relative to oneanother in a parallel, spaced-apart orientation by a plurality of spacerposts 268 disposed at angular intervals around the perimeters of theupper disk 244 and lower disk 256. Each spacer post 268 may be securedin place by means of a suitable fastener, such as a screw, extendingthrough a hole in the upper disk 244 or lower disk 256 and into anopening (e.g. a threaded opening) formed in each end of each of thespacer posts 268.

The receptacle carrier 242 further includes a plurality of receptacledividers 274 extending between each of the outer spokes 264 of the lowerdisk 256 and corresponding outer spokes 252 of the upper disk 244. Thespaces between adjacently disposed receptacle dividers 274 definereceptacle stations 240 each configured to receive a single reaction MRD160. As shown in FIG. 5, which is a bottom plan view of a receptaclecarrier carousel of the incubator, each MRD 160 is carried in agenerally vertical orientation with the lower ends of each receptaclevessel 162 exposed at the bottom of the receptacle carrier 242 and withthe receptacle manipulating structure 166 of each MRD 160 extendingradially beyond the outer perimeter of the receptacle carrier 242.

Details of the receptacle dividers are shown in FIGS. 7-9. As noted,each receptacle divider 274 is attached to one of the outer spokes 264of the lower disk 256, as shown in FIG. 7. The receptacle divider 274includes a divider wall 276 that is oriented generally vertically whenthe divider 274 is installed between the upper disk 244 and lower disk256. The divider wall 276 includes lower positioning posts 278configured to be inserted into mating openings formed in the lower disk256 (not shown) and upper positioning posts 280 configured to beinserted into mating openings (not shown) formed in the upper disk 244.A magnet 282 may be mounted alongside a lower end of the divider wall276. The magnet 282 extends from one edge of the divider wall 276 to anopposite edge. The reaction receptacles carried in the incubator 200 maycontain therein magnetically responsive particles for effecting amagnetic separation procedure for isolating an analyte of interest, andthe presence of such particles within the contents of the reactionreceptacle may obscure any signal emitted by the contents that is to bedetected by the signal detectors 400. Accordingly, the magnet 282mounted to the divider wall 276 aggregates at least a portion of themagnetic particles to one side of the receptacle vessels 162, therebyremoving the particles from a suspension where they can obscure thesignal. More specifically, the magnetic particles used for targetcapture in an embodiment of the invention can affect real-time detectionof amplification products. Two particular interfering effects have beenidentified. First, magnetic particles can inhibit amplification byadsorption of oligonucleotides (e.g., amplification oligonucleotides andprobes) and enzyme reagents (e.g., nucleic acid polymerases). Inaddition, the presence of magnetic particles (settled or in suspension)can result in the dissipation of the fluorescence, thereby blocking orpartially blocking the amount of excitation light that reaches thedetection dyes and the amount of light emitted from the reaction tubes162 of the MRDs 160. This is known as the black cloud effect. Thus, tominimize this effect, the dividers 274 of the incubator 200 include amagnet 282 as shown in FIG. 8.

Each divider 274 includes a receptacle cover flange 284 which will coverthe open upper ends of the receptacle vessels 162, and a receptacle stopwall 286 to prevent the reaction MRD 160 from being inserted into thereceptacle station 240 beyond a desired position. Each divider 274further includes, on one side thereof, a receptacle hanger flange 288and a receptacle guide wing 292, and the opposite side of the dividerwall 276 includes a receptacle support ledge 290. The angled shape ofthe receptacle hanger flange 288 defines a constant spacing between theedge of the hanger flange 288 of one divider 274 and the receptaclesupport ledge 290 of an adjacent divider 274 to provide parallel edgesfor supporting the connecting rib structure 164 of the MRD 160 when theMRD 160 is inserted into the receptacle station 240. The divider 274further includes a receptacle retainer spring 294 configured as aresilient, cantilevered projection with a free end 296 slightly curvedso as to conform to the radially innermost end receptacle vessel 162when the MRD 160 is inserted into the receptacle station 240. Theretainer spring 294, by its own resilience, releasably secures the MRD160 in the receptacle station 240. Each divider 274 further includes adrive belt support element 298 at a lower edge of the divider wall 276.Each drive belt support 298 may include a raised rib 299.

In a preferred embodiment of the invention, the incubator 200 holdseighteen MRDs 160 at a time, each spaced at 20° increments around thecarousel.

A drive assembly 300 of the receptacle carrier 242 includes a motor 302mounted on a motor mount portion 208 of the bottom wall 206 of theincubator housing, guide wheels 304 and 306, and a drive belt 308. Drivebelt 308 is secured around a drive shaft (not shown) of the motor 302,around the guide wheels 304 and 306, and further over the belt drivesupports 298 of the plurality of dividers 274 mounted between the upperdisk 244 and lower disk 256. As noted, each drive belt support 298 mayinclude a vertical rib 299 for engaging the teeth (not shown) of thedrive belt 308. As shown in FIG. 7, which shows a perspective view of abottom wall of the incubator housing, a portion of the receptaclecarrier, and a receptacle carrier drive assembly, the bottom wall 206 ofthe incubator housing includes a plurality of elongated openings 210,preferably formed at equal angular intervals about a point correspondingto the axis of rotation of the receptacle carrier 242. The openings 210are oriented at the same angle at which each MRD 160 will be orientedwhen carried on the receptacle carrier 242, and each opening 210 isconfigured to receive an upper end of a signal detector 400 extendinginto the incubator 200 for detecting signals emitted by the contents ofthe MRDs 160 during the incubation process. Motor 302 is preferably astepper motor under microprocessor control. A “home” position sensor(not shown) indicates when the receptacle carrier 242 is in a specifiedrotational position, and the motor 302 is provided with an encoder.Accordingly, movement of the receptacle carrier 242 can be controlled,e.g., by a microprocessor receiving signals from the home sensor and anencoder coupled to motor 302 to control and monitor the angular movementand positioning of the carrier 242, to sequentially place each MRD 160on the receptacle carrier 242 into a signal detection position above theopenings 210.

As shown in FIG. 10, which shows a partial perspective view ofcomponents of the receptacle carrier of the incubator, the receptaclecarrier 242 further includes a center post 270 extending between theinner ring 258 of the lower disk 256 and the inner ring of the upperdisk 244 (not shown in FIG. 10). A receptacle present sensor 272 ismounted to the center post 270 and is configured to detect the presenceof an MRD 160 inserted into a receptacle station 240 of the receptaclecarrier 242. Microprocessor control, which controls and monitors theangular position of the receptacle carrier 242, also monitors thelocation of each specific MRD (160), which may be identified by, e.g., alabel, such as a machine-readable bar-code or an RFID tag. That is, whenan MRD 160, identified via its label or other means, is moved into theincubator 200, the angular position of the receptacle station 240 intowhich that MRD 160 is inserted is determined and tracked to monitor theposition of that MRD 160 at all times while the MRD is inside theincubator 200.

In one embodiment, the center post 270 and the receptacle present sensor272 are fixed with respect to the receptacle carrier 242. The presenceor absence of an MRD in any receptacle station 240 of the receptaclecarrier 242 can be determined by rotating the receptacle station 240 infront of the receptacle present sensor 272.

Details of the door assembly 214 are shown in FIGS. 4 and 11. The doorassembly 214 includes a door 216 that is slidably disposed in a frame224 secured with respect to a portion of the side wall 202 adjacent thereceptacle opening 204. In FIG. 11, a portion of the frame 224 and theside wall 202 above the opening 204 is omitted so as not to obstructview of the MRD 160. As noted, the door 216 is slidably disposed withinthe frame 224 and may include a groove or ridge along the top and/orbottom edge thereof which engage a ridge or groove along the edges ofthe opening of the frame 224 within which the door 216 is disposed. Agenerally horizontal guide rod 222 spans the opening formed in the frame224 and extends through a lower portion of the door 216. A springmechanism (not shown), such as a coil spring, may be provided to biasthe door 216 into a closed position with respect to the opening 204. Asensor (not shown), such as an optical sensor, may be included toprovide a signal to an instrument controller indicating that the door isa closed and/or opened position.

FIG. 11 also shows a portion of a receptacle transport mechanism 320having a manipulating hook 322 configured to engage the manipulatingstructure 166 of an MRD 160. The receptacle transport mechanism 320includes a housing 324 within which it carries an MRD 160 to and fromthe incubator 200 and/or any other station of an instrument thatincludes the incubator 200. Details of suitable receptacle transportmechanisms are described in more detail at Ammann, et al., “AutomatedProcess For Isolating and Amplifying a Target Nucleic Acid Sequence”U.S. Pat. No. 6,335,166 or Hoerger, et al., “Method and Apparatus forEffecting Transfer of Reaction Receptacles in an Instrument forMulti-Step Analytical Procedures” International Patent Publication No.WO 2010/132885, the contents of which are hereby incorporated byreference. The door assembly 214 includes an actuating post 220extending from the sliding door 216, and a contact element (not shown)of the receptacle transport mechanism 320 engages the actuating post 220to push the sliding door 216 to an open position so that themanipulating hook 322 can be extended through the receptacle opening 204to insert an MRD 160 into the receptacle carrier 242 or to retrieve anMRD 160 from the receptacle carrier 242.

The signal detectors 400 are part of a system that measures, forexample, the concentration of unquenched fluorescent dye molecules inreal time. The assay performed within each receptacle vessel 162 of eachMRD 160 may be preferably designed such that the fluorescent signalincreases as the concentration of target is increased by amplification.The signal detector 400 is used to monitor the amplification process bymonitoring the emergence of the fluorescent signal.

An exemplary embodiment of the incubator 200 may include between threeand six signal detectors 400, where each detector is designed to measurea particular fluorescent dye (i.e., color). Each signal detector 400houses, for example, five individual detectors, which may comprisefluorometers. The five individual fluorometers (also referred to hereinas “channels”) are spaced with the same spacing as that of thereceptacle vessels 162 of each MRD 160. The signal detector 400 may beprovided with additional or fewer individual detectors, but the numberof detectors generally corresponds to the number of receptacle vesselsin each MTU 160. The signal detectors 400 are mounted to theamplification incubator 200 with such an orientation that each of themcan detect signal emitted by each receptacle vessel 162 of an MRD 160when the receptacle carrier 242 stops at preset angular incrementscorresponding to the angular positions of the signal detectors 400.Therefore, each MRD 160 can be scanned by each signal detector 400 onceper revolution of the carrier 242.

As shown in FIG. 12, which is a partial perspective view of a portion ofthe incubator, in one embodiment, six signal detectors 400 areconstructed and arranged to detect signals emitted by the contents ofeach of the five receptacle vessels 162 of six different MRD's 160carried within the housing of the incubator 200. That is, each signaldetector 400 is configured to detect a signal emitted by each of thefive receptacle vessels 162 of an MRD 160 operatively positioned withrespect to the signal detector 400 by the carrier 242. The signaldetectors 400 may be of substantially identical constructions, but eachmay be adapted to detect a signal characteristic of a differentmeasurable or detectable value. For example, each signal detector 400may be configured to detect fluorescence of a different wavelength, andthus each may be configured, or tuned, to detect a different fluorescentdye within the contents of the receptacle vessel 162. Each signaldetector 400 may also be configured to emit light at a predefinedwavelength or within a range of wavelengths. The wavelength of theemitted light from the signal detector 400 frequently corresponds to anexcitation wavelength window of a fluorescent dye within the contents ofthe receptacle vessel 162. The motor 302, which drives the receptaclecarrier 242, is under the control of a microprocessor which may receivesignals from a home sensor coupled to the carrier 242, a timer, and anencoder coupled to the motor 302 for controlling movement and angularpositioning of the carrier 242. The carrier 242 is controlled to (a)move MRDs 160 into operative, sensing positions with respect to thesignal detector(s) 400, (b) pause for a sufficient period of time topermit the signal detector(s) to take and process a signal reading fromthe MRD operatively positioned with respect to it, and (c) index thecarrier 242 to position the next MRD(s) 160 into operative position(s)with respect to the signal detector(s) 400. The signal detectors 400attached to the incubator 200 for real-time fluorescence detection areknown as optical detection modules (a type of a signal detector orsignal measuring device), as will now be described.

Details of a signal detector 400 for use in conjunction with the presentinvention are shown in FIGS. 13-16. As shown in FIG. 13, which is aperspective view of a signal detector, the detector 400 includes ahousing that comprises a detector housing 418 and an excitation housing402, both connected at a right angle with respect to each other to alens and filter, or optics, housing 434. An interface cap 456 isattached to the optics housing 438. Each of the housing components 402,418 and 434 may be made from machined aluminum and secured to oneanother by suitable fasteners, such as screws, and are preferablyanodized. The interface cap 456 is preferably machined fromnon-thermally conductive material, such as Delrin®, so as to minimizethermal conduction between the incubator 200 and the detector 400. Anexcitation printed circuit board (“PCB”) 406 is connected to an end ofthe excitation housing 402, and a detector PCB 422 is connected to anend of the detector housing 418. Excitation and detector circuitrydisposed on the excitation PCB 406 and the detector PCB 422,respectively, are described below. A flexible cable 454 connects theexcitation PCB 406 with the detector PCB 422.

The interface cap 456 includes a rim flange 460 surrounding theperiphery of the cap 456 and a dome portion 458 projecting above the rimflange 460. As shown, for example, in FIG. 12, the dome 458 of theinterface cap 456 extends into the detector opening 210 formed in thebottom wall 206 of the incubator 200, and the rim flange 460 abuts thebottom portion of the bottom wall 206 surrounding the detector opening210 so as to provide a light-tight seal between the interface cap 456and the bottom wall 206. A gasket material may be provided between therim flange 460 and the bottom wall 206 to further enhance thelight-tight seal. Five detection openings 462 are provided in theinterface cap 456.

As shown in FIGS. 15 and 16, which show a side cross-sectional view andan exploded perspective view, respectively, of the signal detector, theexcitation housing 402 includes five excitation channels 404. Anexcitation source 405, such as a light-emitting diode (“LED”) coupled tothe excitation PCB 406 is located at the end of each excitation channel404. Similarly, the detector housing 418 includes five emission channels420, and a detector element 423, such as a photodiode, is provided ineach emission channel 420 and is coupled to the detector PCB 422. Astandoff 406 is mounted between the excitation housing 402 and thedetector PCB 422 at a distance from the detector housing 418 to provideadditional stability for the detector PCB 422.

Within each individual fluorometer, or channel, of each detector 400,there are two optical paths defined by excitation optics and emissionoptics disposed, at least partially, within the excitation and emissionchannels, respectively. As described in more detail below, theexcitation optical path begins with an LED as the light source, whichlight is collimated by an excitation lens and then filtered through anexcitation filter. The filtered light passes upward through a beamsplitter and is focused onto a receptacle vessel 162 by objective lensesbetween the receptacle vessel 162 and the beam splitter. The emissionoptical path originates from the light emitted by the contents of thereceptacle vessel 162, which is collimated by the objective lenses asthe light passes toward the beam splitter and is reflected by the beamsplitter toward the emission channel. Within the emission channel, afterbeing filtered through an emission filter, the light is focused by anemission lens onto the detector element 423, such as a photodetector.

The various optical elements of the detector 400 are located in theoptics housing 434. For each excitation channel 404 of the excitationhousing 402, the optics housing 434 contains excitation optics 408, foreach emission channel 420 of the detector housing 418, the opticshousing 434 contains emission optics 424, and for each detector opening462 of the interface cap 456, the optics housing 434 containsinput/output optics 444. The excitation optics 408, emission optics 424,and input/output optics 444 are disposed within optics channels 436formed within the optics housing 434.

The excitation optics include an excitation lens 412, a lens holder 414,and an excitation filter 416. An O-ring 410 provides a light-tight sealbetween the excitation housing 402 and the optics housing 434. Theexcitation filter 416 is selected so as to pass excitation light fromthe light source 405 within the excitation channel 404 having a desiredexcitation characteristic (e.g., wavelength).

The emission optics include an emission lens 428, a lens holder 430 andan emission filter 432. An O-ring 426 provides a light-tight sealbetween the detector housing 418 and the optics housing 434. Theemission filter 432 is selected so as to transmit only that portion of asignal emitted by the contents of a reaction receptacle to the detector423 within the emission channel 420 having a desired signalcharacteristic (e.g., wavelength).

The input/output optics 444 include a first objective lens 450 and asecond objective lens 448 with a spacer ring 446 disposed therebetween.An O-ring 452 provides a light-tight seal between the interface cap 456and the optics housing 434.

The detector 400 further includes dichroic beam-splitters comprisingdichroic beam-splitter elements 440 held within a beam-splitter frame442 which is inserted into a beam-splitter opening 438 of the opticshousing 434. A beam-splitter 440 is provided for each excitation channel404 and corresponding emission channel 420. The beam-splitter 440 isselected so as to pass excitation light having a prescribed excitationwavelength in a straight optic path from the excitation channel 404 andto deflect emission light from the contents of the receptacle 162 havinga prescribed detection wavelength toward the detection channel 420.

Different fluorescent dyes are excited at different wavelengths. In onemultiplex application of the present invention, suitable dyes includethe rhodamine dyes tetramethyl-6-rhodamine (“TAMRA”) andtetrapropano-6-carboxyrhodamine (“ROX”) and the fluorescein dyes6-carboxyfluorescein (“FAM”) and, each in combination with a DABCYLquencher. Additional suitable fluorescent dyes include6-carboxy-1,4-dichloro-2′,7′-dichloro-fluorescein (“TET”),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy-fluorescein (“JOE”), NED, CALFLUOR Red 610, CAL FLUOR Orange 560, Cy5, QUASAR 670, Texas Red, amongothers. Another suitable dye includes 5′-hexachlorofluoresceinphosphoramidite (“HEX”). A variety of additional fluorescent dyes areknown in the art and can be readily employed in the methods and systemsof the present disclosure. The excitation spectra of the preferred dyesare shown in FIG. 17. Because the preferred dyes are excited atdifferent wavelengths, each detector 400 preferably emits an excitationlight at or near the desired excitation wavelength (i.e., color) for theparticular dye for which the optical detection module is intended.Accordingly, component selection for the optical system will, in manyinstances, be governed by the particular dye for which the detector 400is intended. For example, with respect to the light source 405, theparticular LED selection will depend on the dye for which the detectoris intended.

The detectors 400 are often identical in design and components, with theexception of components that are dye specific. The components that aredye specific include, for example, the light source 405, the excitationfilter 416, the emission filter 432, and the beam splitter 440.

The following table provides specifications for the different filtersfor different types of exemplary dyes:

Filter Specifications

TABLE 1 Center Band- Wavelength width Dimensions Thick- Description (nm)(nm) (mm) ness FAM Excite Filter 460 60 8.9 × 8.9 2 square FAM EmissionFilter 525 30 8.9 × 8.9 2 square FAM Short Wave Pass 10 × 14.8 1.05Dichroic rectangular HEX Excite Filter 535 22 8.9 × 8.9 2 square HEXEmission Filter 567 15 8.9 × 8.9 2 square HEX Short Wave Pass 10 × 14.81.05 Dichroic rectangular ROX Excite Filter 585 29 8.9 × 8.9 2 squareROX Emission Filter 632 22 8.9 × 8.9 2 square ROX Short Wave Pass 10 ×14.8 1.05 Dichroic rectangular

The following table provides specifications for the different lensescorresponding to different types of dyes:

Lens and O-Ring Specifications

TABLE 2 Dye = FAM, HEX, ROX Part No. Description Vendor NT47-475Emission Lens Edmund or Ross NT47-477 Excitation Lens Edmund or RossNT47-476 Objective Lens Edmund or Ross 94115K478 O-ring McMaster

The following table shows preferred characteristics for LED's fordifferent colors:

LED Specifications

TABLE 3 Characteristic Blue Green Amber Chip Size 24 mil 11 mil 25 milDominant Wavelength 462 nm 533 nm 590 nm Radiant Flux 4 mW 2 mW 1.2 mWMax DC forward current 200 mA 50 mA 150 mA

Note that in the illustrated embodiment, the beam splitter 440 passesthe excitation light and reflects the emission light. Since theexcitation channel is longer than the emission channel, this arrangementprovides a narrow profile for the housing of the signal detector 400,thereby maximizing the number of detectors 400 that can be positioned atangular intervals beneath the incubator 200, as shown in FIG. 12.Spatial limitations and preferences may be accounted for in designingthe excitation and emission channels, which can be interchanged from theformat depicted in FIG. 12. In such an embodiment a beam splitter thatreflects the excitation light and passes the emission light could beused.

The process steps of a real-time amplification assay procedure 1900performed in accordance with aspects of the present invention areillustrated in the flow chart shown in FIG. 19. The procedure 1900 isperformed by a diagnostic analyzer of which one or more incubators, suchas incubator 200, is a component and which is controlled by a computer(microprocessor) executing software that includes an algorithm embodyingprocedure 1900 encoded or stored on a computer-readable medium. Theprocess shown in FIG. 19 is similar to an analogous process described indetail in Lair et al., U.S. Pat. No. 8,008,066. The steps describedrepresent exemplary TAA procedures only. Persons of ordinary skill willrecognize that the steps described below may be varied or omitted orthat other steps may be added or substituted in accordance with otherreal-time amplification assay procedures now known or yet to bedeveloped. Reagent formulations for performing a host of amplificationprocedures are well known in the art and could be used in or readilyadapted for use in the present invention. See, e.g., Kacian et al., U.S.Pat. No. 5,399,491; Becker et al., U.S. Pat. No. 7,374,885; Linnen etal., Compositions and Methods for Detecting West Nile Virus, U.S. Pat.No. 7,115,374; Weisburg et al., “Compositions, Methods and Kits forDetermining the Presence of Trichomonas Vaginalis in a Test Sample,”U.S. Pat. No. 7,381,811; and Kacian, “Methods for Determining thePresence of SARS Coronavirus in a Sample,” U.S. Patent ApplicationPublication No. 2010-0279276 A1, the disclosure of each of which isincorporated by reference.

The process steps of an exemplary real-time TAA amplification assaybegin with step 1902, in which a receptacle, such as an MRD 160, ismoved to a pipetting position in a sample transfer station (not shown).In step 1904, a sample pipette assembly (not shown) dispenses 100 μL ofa target capture reagent (“TCR”) including magnetically-responsiveparticles into the receptacle, e.g., into each receptacle vessel 162 ofthe MRD 160. The target capture reagent includes a capture probe, adetergent-containing lytic agent, such as lithium lauryl sulfate, forlysing cells and inhibiting the activity of RNAses present in the samplematerial, and about 40 μg Sera-Mag™ (MG-CM) Carboxylate Modified(Seradyn, Inc., Indianapolis, Ind.; Cat. No. 24152105-050250), 1 micron,super-paramagnetic particles having a covalently bound poly(dT)14. Thecapture probe includes a 5′ target binding region and a 3′ region havinga poly(dA)30 tail for binding to the poly(dT)14 bound to the magneticparticle. The target binding region of the capture probe is designed tobind to a region of the target nucleic acid distinct from the regionstargeted by the primers and the detection probe.

In step 1906, 400 μL of sample is dispensed into the receptacle. In step1908, the receptacle, e.g., MRD 160, is moved to a mixer (not shown),and in step 1909, the sample and TCR are mixed, preferably at 16 Hz for60 seconds. Note that the times given in FIG. 19 and the descriptionthereof are desired times, and the actual times may, in practice, varyfrom the given desired times.

In one embodiment, the diagnostic analyzer includes three incubatorsmaintained at three different temperatures: a first incubator maintainedat 64° C. for target capture and primer annealing, a second incubatormaintained at 43.7° C. for pre-heating receptacles, AT binding, andprimer binding, and a third incubator maintained at 42.7° C. foramplification. The first, second, and third incubators may be configuredthe same as incubator 200 described above, although the first and secondincubators may omit the signal detectors 400.

In step 1910, the receptacle is moved to the second incubator topre-heat the receptacle and its contents at a temperature of 43.7° C.for 276 seconds. In other embodiments, the receptacle may be placed in atemperature ramping station (i.e., a temperature-controlled enclosureconfigured to receive and hold one or more receptacles (not shown)) forthe pre-heating step. In step 1912, the receptacle is moved to the firstincubator (i.e., target capture (“TC”) incubator) where it resides at64° C. for 1701 seconds for hybridization of the capture probe to targetnucleic acids extracted from the sample (though not wishing to be boundby any particular theory, at this temperature, there will be noappreciable hybridization of the capture probe to the immobilizedpoly(dT)14 oligonucleotide.) In step 1914, the receptacle is moved fromthe TC incubator to the second incubator for AT binding where it is heldfor 313 seconds at 43.7° C. to allow for immobilized oligonucleotidesassociated with the magnetic particles to bind to the capture probes. Instep 1916, the receptacle is moved to a cooling chiller (i.e., atemperature-controlled enclosure configured to receive and hold one ormore receptacles (not shown)) where the receptacle is held at 18° C. for481 seconds.

In step 1918, the receptacle is moved to a magnetic parking station (notshown), which is a structure configured to hold one or more receptaclesin proximity to one or more magnets so that the contents of eachreceptacle vessel 162 are exposed to a magnetic field to draw themagnetically-responsive particles of the target capture reagent to aportion of the receptacle adjacent to the magnet and out of suspension.A suitable magnetic parking station is described in Davis, et al., U.S.Patent Application Publication No. 2010/0294047, “Method and System forPerforming a Magnetic Separation Procedure,” the disclosure of which isincorporated by reference.

In step 1920, the receptacle is moved to a magnetic separation station(not shown) for the magnetic separation wash procedure, such as isdescribed in Lair et al., U.S. Pat. No. 8,008,066. Within the magneticseparation station, magnets, which are selectively placed in closeproximity to the reaction vessel, are used to draw and hold themagnetically-responsive particles to a portion of the vessel. Once themagnetically-responsive particles, and any target nucleic acid boundthereto, are thus immobilized, the hybridized nucleic acid can beseparated from non-hybridized nucleic acid by aspirating fluid from thereaction vessel. After the initial aspiration of the fluid contents fromthe vessel, 1 mL of wash solution is added to the receptacle in step1922. Step 1924 comprises a second magnetic wash, which includes, afterthe fluid contents of the receptacle are aspirated, adding 1 mL washsolution to the receptacle in step 1926 and adding 100 μL oil (e.g.,silicone oil), or other surface treating agent, to the receptacle instep 1928. In step 1930, a final magnetic wash procedure is performed(in other embodiments, more or fewer magnetic wash procedures can beperformed) followed by a final dispense of 100 μL oil (e.g., siliconeoil), or other surface treatment agent, in step 1932.

An advantage of adding a surface treating agent, such as silicone oil,to the sample solution in steps 1928 is that it reduces the amount ofmaterial that adheres to the inner surfaces of the reaction vessels 162during the rinsing and aspiration steps of a magnetic separation washprocedure, thereby facilitating a more effective magnetic separationwash procedure. Although the MRDs 160 are preferably made of ahydrophobic material, such as polypropylene, small droplets of material,such as wash solution, may still form on the inner surfaces of the MRDreceptacle vessels 162 during the aspiration steps of a magneticseparation wash procedure. If not adequately removed from the receptaclevessels 162 during the magnetic separation wash procedure, this residualmaterial, which may contain nucleic acid amplification inhibitors, couldaffect assay results. In alternative approaches, the surface treatingreagent could be added to the receptacle vessels 162 and removed priorto adding TCR and sample or the surface treating agent could be added tothe reaction tubes after TCR and sample have been aspirated from thereaction tubes, possibly with the wash solution, and then removed priorto adding amplification and enzyme reagents to the reaction tubes. Theobjective is to provide inner surfaces of the receptacle vessels 162with a coating of the surface treating agent Inhibitors of amplificationreactions are known in the art and depend on the sample source andamplification procedure to being used. Possible amplification inhibitorsinclude the following: hemoglobin from blood samples; hemoglobin,nitrates, crystals and/or beta-human chorionic gonadotropin from urinesamples; nucleases; proteases; anionic detergents such as sodium dodecylsulfate (SDS) and lithium lauryl sulfate (LLS); and EDTA, which is ananticoagulant and fixative of some specimens that binds divalent cationslike magnesium, which, as noted above, is a cofactor used in nucleicacid-based amplification reactions. See, e.g., Mahony et al., J. Clin.Microbiol., 36(11):3122-2126 (1998); Al-Soud, J. Clin. Microbiol.,39(2):485-493 (2001); and Kacian et al., “Method for SuppressingInhibition of Enzyme-Mediated Reactions By Ionic Detergents Using HighConcentration of Non-Ionic Detergent,” U.S. Pat. No. 5,846,701, thedisclosure of each of which is incorporated by reference. Silicone oilis added to each reaction vessel 162 of the MRD 160 in step 1932 toprevent evaporation and splashing of the fluid contents duringsubsequent manipulations.

In step 1934, amplification reagent, which is stored in a chilledenvironment, is added to each receptacle while the receptacle is held at45° C. at an amplification load station (not shown). In step 1936, 75 μLof an amplification reagent are dispensed into the receptacle disposedwithin the load station, and the receptacle is then mixed for 25 secondsat 16 Hz by a mixer incorporated into the load station. For theexemplary TAA reactions, the amplification reagents contain an antisensepromoter-primer having a 3′ target binding region and a 5′ promotersequence recognized by an RNA polymerase, a sense primer that binds toan extension product formed with the promoter-primer, nucleosidetriphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP,including modified nucleotides or nucleotide analogs), and cofactorssufficient to perform a TAA reaction. For the real-time TAAamplification assay, the amplification reagent also contains stranddisplacement, molecular torch probes having interacting label pairs(e.g., interacting fluorescent and quencher moieties joined to the 5′and 3′ ends thereof by conventional means) and a target specific regioncapable of detectably hybridizing to amplification products as theamplification is occurring and, preferably, not to any non-targetnucleic acids which may be present in the receptacles. See Kacian etal., U.S. Pat. No. 5,399,491; Becker et al., “Single-Primer Nucleic AcidAmplification,” U.S. Pat. No. 7,374,885 (disclosing an alternativeTAA-based amplification assay in which an antisense primer and a sensepromoter oligonucleotide blocked at its 3′ end are employed to minimizeside-product formation); and Becker et al., U.S. Pat. No. 6,361,945, thedisclosure of each of which is incorporated by reference.

In step 1938, the receptacle is moved to the second incubator andpreheated at 43.7° C. for 286 sec. In step 1940, the receptacle is movedto the first incubator and incubated at 64° C. for 636 seconds forprimer annealing. In step 1942, the receptacle is moved to the secondincubator and incubated for 405 seconds at 43.7° C. for binding of thepromoter-primer to a target nucleic acid. The preferred promoter-primerin this particular TAA example has a promoter sequence recognized by aT7 RNA polymerase.

In step 1944, the receptacle is moved to the load station for enzymereagent addition at 45° C. In step 1946, 25 μL of enzyme are added andthe MRD is mixed at 10 Hz for 15 seconds. In step 1948, the receptacleis moved to the third incubator (amplification incubator), where thereceptacle contents are incubated at 42.7° C. for 3569 seconds foramplification. During amplification, real-time fluorescence measurementsare taken in step 1950. The enzyme reagent of this example contains areverse transcriptase and a T7 RNA polymerase for performing TAA.

After the nucleic acid-based assay is complete, and to avoid possiblecontamination of subsequent amplification reactions, the reactionmixture can be treated with a deactivating reagent which destroysnucleic acids and related amplification products in the reaction vessel.In such an example, following amplification and real-time measurements,in step 1952, the receptacle is moved to a deactivation queue, or module(not shown), and, in step 1954, 2 mL of a bleach-based agent areprovided to each receptacle to deactivate nucleic acid (i.e., alter thenucleic acid such that it is non-amplifiable) present in the receptacle.Such deactivating agents can include oxidants, reductants and reactivechemicals, among others, which modify the primary chemical structure ofa nucleic acid. These reagents operate by rendering nucleic acids inerttowards an amplification reaction, whether the nucleic acid is RNA orDNA. Examples of such chemical agents include solutions of sodiumhypochlorite (bleach), solutions of potassium permanganate, formic acid,hydrazine, dimethyl sulfate and similar compounds. More details of adeactivation protocol can be found in, e.g., Dattagupta et al., U.S.Pat. No. 5,612,200, and Nelson et al., U.S. Patent ApplicationPublication No. US 2005-0202491 A1, the disclosure of each of which ishereby incorporated by reference.

As noted above, the incubator 200 includes a number of signal detectors400 configured to measure in real time the concentration of unquenchedfluorescent dye molecules located in the MRD 160. As discussed above,the assay is designed such that the fluorescent signal increases as theconcentration of the target is increased by amplification. The detectors400, therefore, may be used to monitor the amplification process bymonitoring the emergence of the fluorescent signal.

As also noted above, incubator 200 may include between 3 and 6 detectors400. Each of the detectors 400 can itself have a number of fluorometers.For instance, according to some embodiments of the present invention,each detector 400 includes five fluorometers, or channels. Preferablythere are as many fluorometers as there are receptacles 162 in the MRD160. Each fluorometer, or channel, includes two optical paths: anexcitation path and an emission path.

Optical crosstalk between adjacent channels of a single signal detector400 and/or between adjacent signal detectors 400 can take a number offorms. Crosstalk can occur when one channel detects the excitation lightfrom another channel (of the same or different signal detector 400) orwhen one fluorometer picks up emission light from a receptacle 162 thatis excited by a different fluorometer. In addition, crosstalk can occurwhen an excitation signal for a particular dye color excites a dye of acolor that is not intended for that fluorometer. For example, as shownin FIG. 17, which shows excitation intensity versus wavelength forseveral different fluorescent dyes, the excitation wavelengths of FAMand JOE, JOE and TAMRA, and TAMRA and ROX overlap to a certain extent.

The data acquisition system and process can be described at a high levelwith reference to FIG. 24. In general, the system and process includethree components: excitation 406, detection 422, and control 506. In theExcitation branch 406, a power source generates an alternating current(“AC”) waveform (e.g., square or sinusoidal) from a voltage-controlledcurrent source and sends that waveform to the light source (e.g., anLED) to generate an excitation light signal that is modulated in amanner corresponding to the AC wave form. The detection branch 422includes a light detector (e.g., a photodiode) that converts photons oflight that impinge on the detector to a current. The detection branch422 further includes a component, such as a transimpedance amplifier,that converts the current from the light detector into a voltage havinga waveform that is the analog of the photons that impinge on the lightdetector. The control branch, or controller, 506 includes, among othercomponents, an analog to digital (A/D) converter and a demodulator. TheA/D converter converts the analog voltage into digital data, and thedemodulator identifies the frequency components of the digital data bydigital signal processing (“DSP”) techniques, such as those describedbelow, and, in particular, identifies the component of the digital datahaving a frequency corresponding to the frequency of the AC waveformthat was used to drive the excitation signal.

Synchronous detection is one means for reducing some forms of crosstalk.Other means for reducing crosstalk include the use of narrow bandwidthspectrum filters for conditioning excitation and emission light,focusing elements (e.g., lenses or narrow apertures) for providing aspatially tight excitation and/or emission beam, and isolating elementsfor optically isolating signal detectors from one another. Synchronousdetection creates a narrow bandwidth frequency filter that is sensitiveto only a narrow range of frequencies in the emission signal centered ata modulation frequency of the excitation signal. In this process, eachphotodetector 423 is connected to a transimpedance amplifier on thedetector PCB 422, and each LED light source 405 is connected to a DACcontrolled current source on the excitation PCB 406. The LED current ismodulated. The signal from the photodiode at the output of thetransimpedance amplifier is demodulated at the modulation frequency ofthe LED forming a synchronous detection system that can rejectcross-talk signals and ambient light.

In addition to cross-talk, synchronous detection also greatly reducesthe noise due to other sources, such as ambient light, allowing the useof an incubator that is not light-tight (i.e., permitting a certainamount of ambient light entry into the interior of the incubator). Usingsynchronous detection, the system is sensitive to signals that are atthe modulation frequency of the excitation signal and is insensitive tosignals that are not at the excitation modulation frequency. Ambientlight is typically not at the modulation frequency of the system and soit is rejected or not measured. For example, sunlight is not modulated;it is DC, so that its intensity is not frequency-dependent. Also,interior room lights are typically modulated at 50, 60, 100, or 120 Hz.Thus, by setting modulation frequencies of the excitation signals awayfrom typical frequencies that would be produced by ambient light, thesystem becomes insensitive to the ambient light.

Crosstalk between emission signals of neighboring receptacles 162 of anMRD 160 can occur because the emission channels 420 and excitationchannels 404 of the signal detector 400 are arranged in close proximityto one-another, e.g., as closely-adjacent channels arranged in a row.Two methods are used to reduce this type of crosstalk. For example, theoptics are designed to have a narrow excitation and detection spot 2 mmin width. The emission optical path of the signal detector 400 acceptslight in a narrow range of angles within this small spot. This reducesthe amount of light that can leak between adjacent receptacles 162 ofthe MRD 160. In addition to the optical means, the synchronous detectionsystem is used to reduce this type of cross talk as well. The modulationfrequency of the excitation signal of each of the five excitationchannels 404 of the signal detector 400 is set at one of twofrequencies, for example, that alternate between adjacent channels. Forexample, the modulation frequency of the excitation signal of the firstchannel corresponding to the first receptacle 162 is set to frequency A,the modulation frequency of the excitation signal of the second channelcorresponding to the second receptacle 162 is set to frequency B, themodulation frequency of the excitation signal of the third channelcorresponding to the third receptacle 162 is set to frequency A, themodulation frequency of the excitation signal of the fourth channelcorresponding to the fourth receptacle 162 is set to frequency B, andthe modulation frequency of the excitation signal of the fifth channelcorresponding to the fifth receptacle 162 is set to frequency A, wherefrequency A and frequency B are set to values that are far enough apartthat each channel is insensitive to its neighbors. Specifically, for theFAM signal detector, the modulation frequency is set to 200 Hz for thefirst, third and fifth receptacles 162 and to 250 Hz for second andfourth receptacles 162.

Crosstalk can also occur between detection of one color and excitationof another color. For example, the FAM emission filter overlaps with theHEX excitation filter such that the excitation light of HEX can bedetected by the FAM fluorometer. This is not the case for the HEXdetector and the FAM excitation, because these two filters do notoverlap. Crosstalk between detection and excitation of adjacent colorsis reduced by two methods. One is to arrange the band pass of thefilters, where possible, so that they don't overlap. As the filterbandwidth is widened, a broader spectrum of light passes through thefilter, and more signal can be obtained, but the amount of cross-talkalso generally increases. Accordingly, the filter bandwidth can beadjusted to provide for a balance between crosstalk and signal strengthto permit detection of a clear emission signal. As shown in FIG. 30,which shows the bandwidths of excitation and emission filters for ROX,HEX, and FAM dyes (for each dye, the excitation bandwidth is to the leftof the emission bandwidth), the spectral bandwidths of FAM and ROXfilters preferably do not overlap. As shown in FIG. 31, which showsnormalized excitation and emission fluorescence versus wavelength forFAM, HEX, and ROX dyes, the HEX excitation wavelength band partiallyoverlaps with FAM emission wavelength band, and the ROX excitationwavelength band partially overlaps with HEX emission wavelength band.See also Table 1 above. For acceptable performance, it is preferablethat the FAM emission and HEX excitation filters overlap because theyare very closely spaced. Typically there is overlap in filters betweencolors that are close, so in the progression of colors from FAM to HEXto ROX, there is overlap between FAM emission and HEX excitation andbetween HEX emission and ROX excitation, but not between FAM (excitationor emission) and ROX (excitation and emission).

Synchronous detection is used to reduce the crosstalk betweenneighboring colors as follows. The alternating of two modulationfrequencies was already described to eliminate crosstalk between theneighboring receptacles 162. In this case, the same method is used; twomodulation frequencies, different from the modulation frequencies usedfor the FAM signal detector, are selected for the HEX signal detectorand are alternated between receptacles 162, and then the FAM modulationfrequencies are used for the ROX signal detector. This arrangement isoutlined below in Table 4:

TABLE 4 Recep- Recep- Recep- Recep- Recep- Dye tacle 1 tacle 2 tacle 3tacle 4 tacle 4 FAM 200 Hz 250 Hz 200 Hz 250 Hz 200 Hz HEX 300 Hz 350 Hz300 Hz 350 Hz 300 Hz ROX 200 Hz 250 Hz 200 Hz 250 Hz 200 Hz

FIG. 25 depicts a logical block diagram of an arrangement of thedetection circuitry 422 according to embodiments of the presentinvention. The detection circuitry on the detector PCB 422 can includedetector circuits 502 a-502 e, which are configured to detectfluorescent light and to convert the detected light to a voltage signalthat can be processed by the controller 506. The output from thedetector circuits 502 a-502 e can be connected to controller 506 eitherdirectly or through a multiplexer 504, as is shown in FIG. 25.

FIG. 27 depicts a logical block diagram of an arrangement of theexcitation circuitry according to embodiments of the present invention.As shown in FIG. 27, which is a block diagram schematically illustratingan arrangement of excitation circuitry embodying aspects of theinvention, excitation circuitry can include the controller 506 and adigital to analog converter (DAC) 510. The excitation circuitry on theexcitation PCB 406 includes excitation circuits 512 a-512 e for drivingeach excitation source 405 of each excitation channel 404. Theexcitation circuits 512 a-512 e are driven by a digital to analogconverter (DAC) controlled current source. The current source is avoltage to current amplifier that controls the current flowing throughthe excitation source 405.

A monitor 516 can be connected to excitation circuits 512 a-512 e tofacilitate process control of the excitation voltage. Checking thevoltage across the LED and the current through the LED give a goodindication if the LED is functioning correctly. This is a diagnosticcapability that can be used in a variety of ways. For example, the LEDcould be checked at power-on, during a self-test, so when thefluorometer powers up it could put a known current through the LED, andif the forward voltage of the LED is in and expected range, then thesystem would pass the self-test. These values could also be checkedduring an assay to monitor correct functioning of the LED.

According to embodiments of the invention, each of the LEDs(corresponding to excitation source 405) in circuits 512 a-512 e can bedriven by a digital to analog converter controlled current source, asshown in FIG. 28, which is a circuit diagram illustrating a fluorometerexcitation circuit. The current source can be a voltage to currentamplifier that controls the current flowing through LED (correspondingto excitation source 405).

In addition to performing the function of driving a computer controlledcurrent waveform through the LED, the current source shown in FIG. 28allows for process control based on LED current and voltage. The outputof the circuit formed by U3 is a monitor of the voltage across the LEDand can be digitized by monitor 516 using an A/D converter. Similarlythe output of R22 (the side away from transistor Q3) can be used tomonitor the current passing through the LED and similarly digitized byan A/D converter located in monitor 516. The current through the LED ismonitored for diagnostic purposes, as described above.

The fluorometer circuits 502 a-502 e could be configured as shown inFIGS. 26A and 26B. Each detector circuit 502 includes a pre-amplifiercircuit, which includes U11, and the amplifier formed by pins 5-7 of U10(designated as U10B). The pre-amplifier circuit receives current fromthe photodiode D (corresponding to detector element 423) and converts itto an amplified voltage. As shown, the amplifier that includes pins 1-3of U10 (designated as U10A) provides a bias current to compensate forthe electrical current out of photodiode D caused by un-modulatedambient light incident on the photodiode D. As with circuitry 1790, thisis to prevent the ambient light from saturating the output of thepre-amplifier.

Amplifiers U11 and U10B form the first two stages of amplification ofthe current signal (corresponding to the emission signal) from thephotodiode D (423). C54, C44, and C58 provide power supplybypassing/filtering to the amplifiers. C12, D12, R55, R57, and R61 forma filtered power supply that biases the anode of the photodiode D.Feedback resistors R31 and R32 convert electrical current from thephotodiode D into a voltage while C48 provides filtering for higherfrequency signals. The voltage divider formed by R43 and R45 provides avoltage gain of 10 in the next pre-amplification state while capacitorC56 provides additional low pass filtering.

The detector circuits 502 a-502 e are configured to use a level shifterformed by U7A. Though not wishing to be bound by theory, the purpose ofthe level shifter is to move the zero level of the pre-amp up to themiddle range of a unipolar analog to digital converter. This allows theuse of A/D converters employed by certain microcontrollers so that anadditional A/D converter is not required.

The circuit configuration shown in FIGS. 26A and 26B does not require ademodulator circuit. Instead, the detection circuitry of the detectorPCB 422 can employ five identical preamplifier circuits on thecontroller board. The output of each of the preamplifier circuits canthen connect directly to the A/D converter input of the controller 506.This allows synchronous detection to be accomplished by DSP using analgorithm executed in the microcontroller 506, instead of using ananalog demodulator and filter circuit as in synchronous detectioncircuits.

According to some embodiments, the signal from each of the detectorcircuits 502 a-502 e is digitized at a rate that is at least twice themodulation frequency of the excitation source 405, preferablysubstantially more than twice the modulation frequency. For instance, inone implementation, the modulation frequency is around 250 Hz, and thedigitization rate is 4 kHz. According to some embodiments, themicrocontroller 506 identifies frequency components of an emissionsignal by executing a Goertzel algorithm modified as described below.The amplitude of the frequency component that matches the excitationmodulation frequency can then be calculated, thus identifying theemission signal that is due to the intended excitation signal anddigitally “filtering” out signal components due to cross-talk andambient light.

In other embodiments, however, the digitized signal is processed by anyof a number of different algorithms for identifying the amplitude of aspecific frequency component, including discrete Fourier transform(DFT), Fast Fourier transform (FFT), or digital lock-in detection.

The Goertzel algorithm employed is a digital signal processing techniquefor identifying frequency components of a signal, published by Dr.Gerald Goertzel in 1958. While the general Fast Fourier transformalgorithm computes evenly across the bandwidth of the incoming signal,the Goertzel algorithm detects frequencies in a single frequency bandand is more efficient than the FFT. The modified Goertzel Algorithmcalculates the frequency component using the last 2 terms of the filterand normalizes the output to be independent of the number of samples.

In embodiments of the invention that employ the modified Goertzelalgorithm, the power of a signal at a certain frequency can becalculated according to method 550 depicted in FIG. 23, which can beimplemented by means of software stored on a computer-readable mediumand executed by a microprocessor (i.e., a programmed computer), such ascontroller 506. According to method 550, several variables areinitialized at step 552. Fs is initialized to the frequency at which thesignal is sampled (i.e., the digitization frequency). Ts is set to thesampling period, or 1/Fs. Fc is set to the center frequency of thefilter in Hz (i.e., the modulation frequency for excitation of theparticular dye being detected). Ti is initialized to an integrationtime. Preferably, a center frequency Fc is chosen that is an integermultiple of the sampling period Ts, and an integration time Ti is chosenthat is an integer multiple of the center frequency Fc, since it hasbeen found that choosing such center frequencies and integration timesminimizes digital processing noise. Finally, the Goertzel terms Q₀, Q₁,and Q₂ are set to zero.

At step 554, a filter coefficient K is calculated using the followingformula:K=2 cos(2πF _(c) /F _(S))

With the filter coefficient K calculated, at step 556 the number ofmeasurements in the integration period N can then be calculated bydividing the integration period Ti by the sampling period Ts, and the“COUNT” variable can be set to zero at step 558. Step 560 pauses theprocess until the next sample time has elapsed. At the next sample time,the analog output from the detector circuit 502 is converted to adigital input Vin at step 562. At step 564, the Goertzel terms arerecalculated in the following order:Q ₀ =KQ ₁ −Q ₂ +VinQ ₂ =Q ₁Q ₁ =Q ₀

That is, first Q₀ is calculated, then Q₂ is set to the current value ofQ₁, and then Q₁ is reset to the newly calculated value of Q₀.

Since the basic Goertzel Algorithm is essentially a discrete Fouriertransform (“DFT”) that produces a sequence of terms related to a singlefrequency of interest, the algorithm is “modified” in the currentimplementation by the following steps:

The counter is incremented at step 566, and it is determined whether Nmeasurements have been taken at step 568. If N measurements have notbeen taken, then the method loops back to step 560. If N measurementshave been taken, then the power P of the signal can be calculated atstep 570 from the last 2 terms (Q1 & Q2) at the end of the samplingperiod according to the following formula:P=√{square root over (Q ₁ ² +Q ₂ ² −KQ ₁ Q ₂)}

At this point, the calculation of the power output can be normalized bydividing P by the number of measurements taken at step 572.

As explained below, processing of the signal depends on measurement ofthe actual linear power. Without these modifications, the output is notlinear and is also dependent on the number of samples. The number ofsamples is a configurable parameter and is not the same for all channelsin all signal detectors.

FIG. 29 is a timing diagram 580 of the movement of the MRD 160 by thecarrier 242 and MRD interaction with each detector 400. As can be seen,the total MRD movement time 582 (e.g., 2 seconds) for each detector 400is divided into several components. Referring to the timing blocksindicated at 584, according to some embodiments, a period (e.g., 0.8second (800 msec)) is specified for moving the receptacle carrier 242 toplace the MRD 160 in an operative position with respect to the signaldetector 400, a slosh period (e.g., 0.8 second (800 msec)) is specified,and a measurement period (e.g., 400 ms) is specified. The slosh timedelay is provided to allow the fluid within the receptacle 162 to stopsloshing (following the starting and stopping of the receptacle carrier242) and to allow any mechanical vibrations to damp out before anemission signal measurement is taken. The measurement period is dividedinto measurement timeslots 586 of 250 μsec duration, and each timeslotis subdivided into 50 μsec timeslots 588 for each of the five channelsof the signal detector 400 corresponding to the five receptacles 162 ofthe MRD 160. Thus, each of the channels is sampled every 250 μsec duringthe 400 ms measurement period, resulting in 1600 samples(0.400/0.00025). These 1600 samples are averaged to provide a singledata point for the 400 ms measurement period.

The times are configurable. For example, a 700 ms slosh period and a 200ms measurement period may be used. A 200 ms measurement period wouldresult in 800 samples for a 250 μsec measurement timeslot, and the 800samples would be averaged to provide a measurement data point for the200 ms measurement period.

Each 50 μsec timeslot 590 for each channel N is divided into differenttasks. During an initial time period 591 (e.g., 1 μsec) the detector 400reads an analog to digital converter (“ADC”) buffer to get the result ofthe previous A/D conversion. More specifically, during each 50 μsectimeslot 590, a different channel is sampled: channel 1 (i.e., a firstreceptacle vessel 162, See FIG. 1) is sampled during one 50 μsectimeslot 590, then channel 2 is sampled during the next 50 μsec timeslot590, then channel 3 is sampled during the next 50 μsec timeslot 590,then channel 4 is sampled during the next 50 μsec timeslot 590, and thenchannel 5 is sampled during the next 50 μsec timeslot 590. In each 50μsec time slot, one of the channels is sampled and one of the channelsis processed. So, for example, when sampling channel 2, channel 1 isprocessed, when sampling channel 3, channel 2 is processed, thenchannels 4 and 3, then channels 5 and 4, then channels 1 and 5, etc. Asexplained above, for each channel, the signal detector converts photonsto an analog voltage, and that voltage is digitized by the A/Dconverter. The digitized voltage is temporarily stored in the ADCbuffer. As the signal detector begins the process for measuring theoptical signal (and converting the signal to digital data), the digitalvalue for the previous channel that is stored in the ADC buffer is readfrom the buffer and the value from the previous channel will be replacedin the ADC buffer by the value derived for the next channel.

This parallel sampling and processing saves time. The data previouslyconverted for the previous channel is read, and then a conversion oncurrently measured data of the next channel begins. While the A/Dconversion of the voltage analog corresponding to the detected opticalsignals proceeds on the present channel, data from the previousconversion and the previous channel, which was read from the ADC buffer,is processed by DSP to identify the component having the correctexcitation frequency. Thus, processing and measurement overlap.

Returning to FIG. 29, during a subsequent time period 592 (e.g., 1 μsec)after reading the ADC buffer, the correct channel N is multiplexed, oractivated, and the A/D cycle begins for that channel. There is an analogmultiplexer that enables selection of one of N analog signals to berouted to the input of the A/D converter. This step selects one of thefive channels in the signal detector 400 (since there is one channel foreach receptacle 162 of the MRD 160 in each color) and starts an analogto digital conversion on that channel. The signal is measured by atwo-step process. During the sampling time, block 593 (e.g., 6.2 μsec),the analog signal is connected by the multiplexer to a sample and holdamplifier. The sampling time is necessary for the sample and holdamplifier to settle to an accurate representation of the analog signal.After the sampling time, the A/D conversion is started, block 594 (e.g.,2.8 μsec). During conversion, the analog signal is disconnected from thesample and hold amplifier, and the voltage stored by the sample and holdamplifier is converted into digital bits by the A/D convertor. Thus, thesignal measured during sampling time slot 593 is converted to a digitalmeasurement of voltage during the conversion time slot 594 and is storedin the ADC buffer register, replacing the previously-stored value, whichis then read in the beginning of the next 50 μsec time slot

The processor calculates a digital value which is a representation ofthe next current data point in the LED current waveform. In parallelwith time blocks 593 and 594, during time block 595 (e.g., 9 μsec), thenext LED current data point in the AC waveform can be sent to a digitalto analog converter (“DAC”) using, for instance, a serial peripheralinterface (SPI) for generating the excitation signal point for thechannel N. The current waveform can be a sine wave, square wave, orother shape. This is configurable and both sine and square waves havebeen used with similar performance.

During time block 596 (e.g., 15 μsec), the digital signal processing(“DSP”) (using, for instance, the modified Goertzel algorithm describedabove) is performed to calculate the power from the data read from theADC buffer during time block 591.

According to some embodiments of the invention, a time block 597 (e.g.,24 μsec) may be unused during each channel's 50 μsec timeslot, which maybe used for performing other tasks.

Once the data has been collected by measuring fluorometric emissionsfrom each receptacle at prescribed intervals for a prescribed period oftime, the data is processed to determine the concentration of aparticular analyte (e.g., target nucleic acid) in the sample. Themeasured data, that is, the measure signal, will be referred to in termsof a Relative Fluorescent Unit (“RFU”), which is the signal generated bythe detection PCB 422 of the signal detector 400 based on the amount ofemission fluorescence focused onto the detection element 423. Each datapoint, measured at a given time interval, is RFU(t). Plots of RFU(t) fora variety of data sets, known as “growth curves” are shown in FIG. 21.In general, each RFU(t) plot is generally sigmoidal in shape,characterized by an initial, flat portion (known as the “static level”or “baseline phase”) at or near a minimum level, followed by an abruptand relatively steeply sloped portion (known as the “growth phase”), andending with a generally flat portion at or near a maximum level (knownas the “plateau phase”).

As used herein, a “growth curve” refers to the characteristic pattern ofappearance of a synthetic product, such as an amplicon, in a reaction asa function of time or cycle number. A growth curve is convenientlyrepresented as a two-dimensional plot of time α-axis) against someindicator of product amount, such as a fluorescence measurement—RFU(y-axis). Some, but not all, growth curves have a sigmoid-shape. The“baseline phase” of a growth curve refers to the initial phase of thecurve wherein the amount of product (such as an amplicon) increases at asubstantially constant rate, this rate being less than the rate ofincrease characteristic of the growth phase (which may have a log-linearprofile) of the growth curve. The baseline phase of a growth curvetypically has a very shallow slope, frequently approximating zero. The“growth phase” of a growth curve refers to the portion of the curvewherein the measurable product substantially increases with time.Transition from the baseline phase into the growth phase in a typicalnucleic acid amplification reaction is characterized by the appearanceof amplicon at a rate that increases with time. Transition from thegrowth phase to the plateau phase of the growth curve begins at aninflection point where the rate of amplicon appearance begins todecrease. The “plateau phase” refers to the final phase of the curve. Inthe plateau phase, the rate of measurable product formation issubstantially lower than the rate of amplicon production in thelog-linear growth phase, and may even approach zero.

A process for calculating an analyte concentration is shown by means ofa flow chart in FIG. 20. The RFU(t) data from the signal detector 400 isinput as represented at box 2100. The RFU(t) data is demodulated by thedetector 400, for example, using the Goertzel algorithm described above.The RFU(t) data goes to threshold time determination, which begins at2104. Threshold time, or T-time, (also known as time of emergence)refers to the time at which the data RFU(t), normalized as discussedbelow, reaches a predefined threshold value. Using calibration curves,as will be described in more detail below, the T-time determined for aparticular sample can be correlated with an analyte concentration,thereby indicating the analyte concentration for the sample. In general,the higher the concentration of the analyte of interest, the sooner theT-time is reached.

The first step of the T-time determination procedure is backgroundadjustment and normalization of the data, as represented at box 2106.Background adjustment is performed to subtract that portion of thesignal data RFU(t) that is due to background “noise” from, for example,stray electromagnetic signals. That is, the background noise includesthat part of the RFU(t) signal due to sources other than the analyte ofinterest. Background adjustment is performed by subtracting a backgroundvalue “BG” from the data RFU(t) to obtain adjusted data RFU*(t). Thatis, RFU*(t)=RFU(t)−BG.

The background BG can be determined in a number of ways.

In accordance with one method for determining the background noise, thefirst step is to determine the time intervals between data points. Thetime interval is determined by multiplying cycle time (i.e., the timebetween consecutive data measurements) by the data point (i.e., 0^(th)data point, 1^(st) data point, 2^(nd) data point, . . . , n^(th) datapoint) and divide by 60 seconds. For example, assuming a cycle time of30 seconds, the time interval for the 15^(th) data point is (15×30sec.)/60 sec.=7.5.

The next step is to find the midpoint of the signal data by adding theminimum signal data point and the maximum signal data point and dividingby two. That is: (RFU_(max)+RFU_(min))/2

Starting at the time corresponding to the midpoint value and workingbackwards, calculate the slope for each pair of data points:(RFU(t)−RFU(t−1))/Δt(t→t−1).

Next, determine where the slope of RFU(t) flattens out by finding thefirst slope value that is less than the static slope value (i.e., thevalue before the RFU(t) curve begins its upward slope). A representativestatic slope value, also known as the “delta value,” includes 0.0001.Once this slope is found, find the next cycle in which the slope that isnot negative or is, for example, above the negative delta value (i.e.,−0.0001); this value is H_(index). Next, take the mean of the entirerange of RFU(t) values starting at the first data point and go to theRFU value that corresponds to the H_(index) value. The mean of this datamay be computed using the Excel TRIMMEAN function on this range of datausing a static back trim value of 0.15 (that is, the lowest 7.5% of RFUvalues in the specified range and the highest 7.5% RFU values in thespecified range are excluded). This mean value is the background, BG.Alternatively, the background can be determined in accordance with theprocedure described above using a delta value other than 0.0001.

A further alternative method for determining the background eliminatesthe delta value criterion and instead take a TRIMMEAN mean of the RFUdata from cycle 1 to a prescribed end point, such as the first cyclebefore 5.5 minutes. For this alternative, the static back trim value maybe adjusted to, for example, 0.40 (that is, the lowest 20% of RFU valuesin the specified range and the highest 20% RFU values in the specifiedrange are excluded from the background calculation).

A further alternative method for determining the background is toperform a curve fit on all or a portion of the RFU data to derive anestimate of the baseline value, which is the background to besubtracted. Any curve fit technique suitable for fitting a curve to theRFU data can be used.

An exemplary curve fit technique is to use a portion of the equationderived by Weusten et al. for curve fit of the typically sigmoidalcurves associated with nucleic acid amplification. See Weusten et al.,Nucleic Acids Research, 30(6e26):1-7 (2002), the disclosure of which isincorporated by reference. For background subtraction, it is onlynecessary to ascertain the baseline level. Thus, it is also onlynecessary to fit a curve to the first portion of the RFU dataencompassing the baseline, usually toward the beginning of the curve.

The curve fit may be performed on the RFU(t) data from cycle 1 to thecycle just before 75% of the maximum RFU. The following polynomialequation (3), which, as mentioned above, is a portion of the equationderived by Weusten et al, is used to generate a best fit model of theRFU data:RFU(t)=Y0+a1a2[e ^(a2(t−a3))/(1+e ^(a2(t−a3)))] ln(1+e ^(a2(t−a3)))  (3)

Initial estimates for the variables Y0, a1, a2, and a3, as discussedbelow, are input to the curve-fit equation and an iterative solutionfitting the equation to the RFU data is performed, for example, usingthe SOLVER function of Microsoft EXCEL, to yield the final equation andthe final values for Y0, a1, a2, and a3.

Y0=is the baseline; an initial value can be RFU(1).

a1=relates to the steep portion (growth phase) of the RFU(t) data; 0.05can be a suitable initial estimate for a1.

a2=relates to the steep portion (growth phase) of the RFU(t) data; 1.0can be a suitable initial estimate for a2.

a3=relates to the transition between the baseline and the slope feature;the time, or cycle, at which RFU(t) reaches a value just before 25% ofRFU_(max) is a suitable initial estimate for a3.

When the final values of Y0, a1, a2, and a3 have been derived, Y0 istreated as the back ground, and is subtracted from the RFU(t) data forwhich the curve fit was performed.

Curve fit equations other than that described above can be used. Forexample, the commercially available TABLECURVE software package (SYSTATSoftware Inc.; Richmond, Calif.) can be used to identify and selectequations that describe exemplary real-time nucleic acid amplificationcurves. One such exemplary resulting equation, used for mathematicalmodeling, is given by equation (4):RFU(t)=Y0+b(1−exp(−(t−d*ln(1−2{circumflex over( )}^((−1/e)))−c)/d)){circumflex over ( )}^(e)  (4)Still another exemplary resulting equation is given by equation (5):RFU(t)=Y0+b/(1+exp(−(t−d*ln(2{circumflex over( )}^((1/e))−1)−c)/d)){circumflex over ( )}^(e)  (5)

In each case, as described above, the equation can be solved, forexample, using the SOLVER function of Microsoft EXCEL, to yield thefinal equation and the final values for Y0 and the other parameters, andthe solutions yields a Y0 that is the background to be subtracted fromthe RFU(t) data.

To normalize the data, each data point, adjusted for the background, isdivided by the maximum data point, also adjusted for the background.That is:

$\begin{matrix}{{{Normalized}\mspace{14mu}{RFU}} = {{RFU}_{n}(t)}} \\{= \frac{{RFU}^{*}(t)}{{RFU}_{\max}^{*}}} \\{= \frac{\left( {{{RFU}(t)} - {BG}} \right)}{\left( {{RFU}_{\max} - {BG}} \right)}}\end{matrix}$Thus, the RFU_(n)(t) will be from −1 to 1.

In step 2108, the range of data is calculated by subtractingRFU_(n(min)) from RFU_(n(max)). If the calculated range does not meet orexceed a specified, minimum range (e.g., 0.05), the data is consideredsuspect and of questionable reliability, and, thus, the T-time will notbe calculated. The minimum range is determined empirically and may varyfrom one fluorescence measuring instrument to the next. Ideally, thespecified minimum range is selected to ensure that the variation of datavalues from minimum to maximum exceeds the noise of the system.

In step 2110, a curve fit procedure is applied to the normalized,background-adjusted data. Although any of the well-known curve fitmethodologies may be employed, in a preferred embodiment, a linear leastsquares (“LLS”) curve fit is employed. The curve fit is performed foronly a portion of the data between a predetermined low bound and highbound. The ultimate goal, after finding the curve that fits the data, isto find the time corresponding to the point at which the curveintersects a predefined threshold value. In the preferred embodiment,the threshold for normalized data is 0.11. The high and low bounds aredetermined empirically by fitting curves to a variety of control datasets and observing the time at which the various curves cross the chosenthreshold. The high and low bounds define the upper and lower ends,respectively, of the range of data over which the curves exhibit theleast variability in the times at which the curves cross the giventhreshold value. In the preferred embodiment, the low bound is 0.04 andthe high bound is 0.36—See FIG. 21. The curve is fit for data extendingfrom the first data point below the low bound through the first datapoint past the high bound.

At step 2110, determine whether the slope of the fit is statisticallysignificant. For example, if the p value of the first order coefficientis less than 0.05, the fit is considered significant, and processingcontinues. If not, processing stops. Alternatively, the validity of thedata can be determined by the R2 value.

The slope m and intercept b of the linear curve y=mx+b are determinedfor the fitted curve. With that information, T-time can be determined atstep 2104 as follows:

${T\text{-}{time}} = \frac{{Threshold} - b}{m}$The technique of using the fitted curve to determine T-times isillustrated graphically in FIG. 22.

Returning to FIG. 20, at step 2116, it is determined whether or notinternal control/calibrator adjustments are desired. Typically, a testprocedure would include at least one reaction vessel with a knownconcentration of a nucleic acid (other than a nucleic acid of interest)as a control, or, alternatively, a control nucleic acid sequence can beadded to each sample. The known concentration can be simply used ascontrol to confirm that a reaction did take place in the reactionvessel. That is, if the known concentration is amplified as expected,successful reaction is confirmed and a negative result with respect tothe target analyte is concluded to be due to absence of target in thesample. On the other hand, failure to amplify the known concentration asexpected indicates a failure of the reaction and any result with respectto the target is ignored.

The known concentration can be used to calibrate the concentration ofthe target. The T-times corresponding to a series of standardscontaining internal control and target sequences are determined for astatistically valid number of data sets. Using this data, a calibrationplot is constructed from which the test sample's concentration isinterpolated as described below.

One method of constructing the calibration plot places the knownconcentrations of target analyte on the x-axis versus the differencebetween target and control T-times on the y-axis. Subsequently, the testsample's concentration is interpolated from the calibration curve fit.Another method of constructing the calibration plot places the knownconcentration of target analyte on the x-axis versus the fraction(target T-time/internal control T-time) on the y-axis. Subsequently, thetest sample's concentration is interpolated from the calibration curvefit. An example of this is disclosed in Haaland, et al., “Methods,Apparatus and Computer Program Products for Determining Quantities ofNucleic Acid Sequences in Samples Using Standard Curves andAmplification Ratio Estimates,” U.S. Pat. No. 6,066,458, the disclosureof each of which is incorporated by reference. A further alternativemethod of constructing the calibration plot utilizes a parametriccalibration method, such as the method described in Carrick et al.,“Parametric Calibration Method,” U.S. Pat. No. 7,831,417, the disclosureof which is incorporated by reference.

Occasionally, data sets exhibit a dip just after the initial staticbaseline (i.e., the initial, flat part of the RFU(t) curve, see FIG. 21)and just before the data begins its upward slope. To identify andcorrect such data, and prior to determining the T-time for that data,the following algorithm is employed. Starting at Hindex, check eachRFU(t) value to determine if it is less than the background value, BG.If yes, subtract RFU(t) from BG (the result should be a positivenumber). This will be the CorValue. Add the CorValue to the backgroundsubtracted value, this in turn will bring RFU(t) up to the baseline.Perform this analysis working forward on each RFU(t) value until thelatest CorValue is less than the preceding CorValue. Add the greatestCorValue to each of the remaining background subtracted RFU(t) values.Now, the corrected data set can be normalized and the T-time determinedas described above.

If a curve fit method is used to derive the background level, it may notbe necessary to perform the dip correction described above. It may alsobe desirable to perform outlier detection on the data set to identifyand, if necessary, discard data points that exhibit abnormal values ascompared to the remaining data points. Any of the well-known outlierdetection methodologies can be used.

The quantitation procedure 2120 is the second part of the analyteconcentration determination. T-times are determined for knownconcentrations of analytes for known conditions. Using this data,relationships between analyte concentrations (typically expressed as logcopy) and T-times can be derived. After a T-time is determined for aparticular sample, the derived relationship (Log copy=f(T-time)) can beused to determine the analyte concentration for the sample.

More specifically, at steps 2122 and 2124, calibration/control data setsfor a control analyte of known concentrations are validated by, forexample, outlier analysis and/or any other known data validationmethodologies. If the data is found to be valid, calibration continues,otherwise, calibration stops.

T-times for the control data sets are determined, and T-time vs. Logcopy is plotted for all samples of a particular condition (e.g., samplesprocessed with reagents from a particular batch lot). In step 2126, acurve fit, such as a linear least squares fit, is performed on a portionof the T-time vs. Log copy plot to find the slope m and intercept b ofthe line that best fits the data. If the number of available T-time vs.Log copy data points (known as “calibrators”) is not less than apredefined minimum number of calibrators (as determined at step 2128),lowest calibrators, if any, are removed at step 2130, as follows:

After finding the best fit line for the calibrator data points, 2^(nd)and 3^(rd) order curve fits are tested as well. If these fits aresignificantly better than the 1st order, linear fit, the calibrator datapoint that is furthest from the linear curve fit is discarded, and1^(st), 2^(nd), and 3^(rd) fits are found and compared again with theremaining calibrators. This process is repeated—assuming that the numberof calibrators is not less than the minimum acceptable number ofcalibrators—until the 2nd and 3rd order fits are not significantlybetter than the 1^(st) order, linear fit.

When the linear T-time vs. Log copy equation has been derived, theconcentration (as Log copy) of the analyte of interest for a sample isdetermined, at step 2132, by plugging the T-time for that sample intothe equation. Thus, the assay results are obtained 2134.

Contemplated enhancements of the RT incubator 608 include self-checkingoptical detection modules. In such a module, a known, standardexcitation signal is emitted by the LED 1732 (or, alternatively, aseparate, dedicated LED) and the excitation light is directed to thephoto diode 1780 (and/or a separate, dedicated comparator photo diode)to ensure that the excitation signals, emission signals, and the signaloutput of the printed circuit board 1790 are all correct.

All documents referred to herein are hereby incorporated by referenceherein. No document, however, is admitted to be prior art to the claimedsubject matter.

While the invention has been described in connection with what arepresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not to be limited to thedisclosed embodiments, but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

Furthermore, those of the appended claims which do not include languagein the “means for performing a specified function” format permittedunder 35 U.S.C. § 112(¶6), are not intended to be interpreted under 35U.S.C. § 112(¶6) as being limited to the structure, material, or actsdescribed in the present specification and their equivalents.

The invention claimed is:
 1. A method for determining a concentration ofan analyte of interest in a sample contained in a receptacle bydetecting an optical signal emitted from the contents of the receptaclein the presence of multiple, different optical signals originated fromthe same receptacle or from different receptacles, said methodcomprising: (a) forming amplification products from the analyte ofinterest contained within the receptacle; (b) hybridizing a detectablylabeled probe to the amplification products; (c) generating an opticalexcitation signal, wherein the optical excitation signal has apredetermined excitation wavelength that excites an emission moiety ofthe probe, which emits an optical emission signal that is associatedwith the excitation wavelength, and wherein the optical excitationsignal is modulated at a predetermined modulation frequency; (d) whileforming the amplification products, directing the optical excitationsignal at the contents of the receptacle; (e) with a signal detectingdevice, detecting an optical signal including an optical emission signalemitted from the contents of the receptacle and converting the detectedoptical signal to an analog detection signal; (f) digitizing the analogdetection signal using a computer-implemented signal-digitizingalgorithm to generate digitized detection data; (g) determining theamplitude of the digitized detection data that is at the predeterminedmodulation frequency by mathematically processing the digitizeddetection data using a computer-implemented digital signal processingtechnique for determining the amplitude of the optical signal at thepredetermined modulation frequency to thereby ascertain the portion ofthe detected optical signal that corresponds to the associated opticalemission signal; and (h) determining the concentration of the analyte ofinterest present within the receptacle by executing acomputer-implemented algorithm for processing data relating to theportion of the detected optical signal that corresponds to theassociated optical emission signal determined in step (g).
 2. The methodof claim 1, further comprising determining a concentration of a secondanalyte of interest in a sample contained in a receptacle by detecting asecond optical signal emitted from the contents of the receptacle by:(i) generating a second optical excitation signal, wherein the secondoptical excitation signal has a predetermined second excitationwavelength that excites an emission moiety of the probe, which emits asecond optical emission signal that is associated with the secondexcitation wavelength and wherein the second optical excitation signalis modulated at a predetermined second modulation frequency; (j) whileforming the amplification products, directing the second opticalexcitation signal at the contents of the receptacle; (k) with the sameor a different signal detecting device, detecting a second opticalsignal including an optical emission signal emitted from the contents ofthe receptacle and converting the detected second optical signal to asecond analog detection signal; (l) digitizing the second analogdetection signal using a computer-implemented signal-digitizingalgorithm to generate second digitized detection data; (m) determiningthe amplitude of the second digitized detection data that is at thepredetermined second modulation frequency by mathematically processingthe second digitized detection data using a computer-implementedsignal-processing technique for determining the amplitude of the secondoptical signal at the predetermined second modulation frequency tothereby ascertain the portion of the second detected optical signal thatcorresponds to the associated second optical emission signal; and (n)determining the concentration of the second analyte of interest presentwithin the receptacle by executing a computer-implemented algorithm forprocessing data relating to the portion of the second detected opticalsignal that corresponds to the associated second optical emission signaldetermined in step (m).
 3. The method of claim 2, wherein the modulationfrequencies are different for at least two optical signals to bedetected.
 4. The method of claim 1, wherein the optical emission signalis a fluorescent emission.
 5. The method of claim 1, wherein theemission moiety comprises a fluorescent dye.
 6. The method of claim 1,wherein step (g) comprises executing a Goertzel signal processingtechnique.
 7. The method of claim 1, wherein the analog detection signalis digitized at a frequency that is at least twice the modulationfrequency of the associated optical excitation signal.
 8. The method ofclaim 7, wherein the modulation frequency is within the range of 200-350Hz, and the digitization frequency is about 4 kHz.